Pharmaceutical composition for treatment of spinocerebellar ataxia

Abstract

Provided is a nucleic acid molecule having an antisense effect against a transcript of a mutated gene that includes an abnormal repeat region.　The nucleic acid molecule has an antisense effect against a transcript of a mutated gene that includes an abnormal repeat region. The abnormal repeat region comprises a 5' region, a core repeat region, and a 3' region. The core repeat region comprises a base sequence in which the base sequence TGGAA repeats multiple times, or a base sequence that is complimentary thereto. The nucleic acid molecule includes a base sequence that is complimentary to four or more continuous bases in the abnormal repeat region of the transcript and includes a target-binding region that can hybridize to the transcript.

Description

Pharmaceutical composition for treating spinocerebellar ataxia

[0001] The present invention relates to nucleic acid molecules that have an antisense effect on the transcripts of mutant genes containing abnormal repeat regions, and pharmaceutical compositions for treating spinocerebellar ataxia.

[0002] Spinocerebellar ataxia type 31 (SCA31) is a dominantly inherited spinocerebellar degeneration. In SCA31, the superior surface of the cerebellar vermis is damaged, and cerebellar Purkinje cells in particular degenerate and are lost.

[0003] SCA31 is a slowly worsening disease that begins with unsteadiness and gradually leads to the inability to walk, and requires the use of a cane or wheelchair within about 10 years, although this varies from person to person. SCA31 is one of the most common spinocerebellar degenerations in Japanese people, with an estimated 2,000 to 3,000 patients in Japan.

[0004] SCA31 was first discovered in 2000 in a family linked to the long arm of chromosome 16 (Non-Patent Document 1). Furthermore, in 2009, it was reported that SCA31 is caused by an abnormal repeat region containing a 2.5-3.8 kb pentanucleotide repeat in an intron shared by two genes, BEAN1 (brain expressed associated with NEDD4; NEDD4-associated brain expression 1) and TK2 (thymidine kinase 2), which are located in the same region but in opposite orientations (Non-Patent Document 2). This pentanucleotide repeat is expressed as a UGGAA repeat in the BEAN1 transcript and as a UUCCA repeat in the TK2 transcript. In SCA31 patients, RNA molecules transcribed from these repeats aggregate within the nucleus. These RNA foci are thought to cause spinocerebellar ataxia.

[0005] Spinocerebellar ataxia type 10 (SCA10) is a spinocerebellar ataxia with a similar molecular pathology. The mutant ataxin-10 (ATXN10) gene in SCA10 has four known alleles, of which allele B transcribes an abnormal repeat region containing an AUUCC repeat, resulting in the formation of RNA foci (Non-Patent Document 3). The AUUCC repeat in SCA10 is identical to the UUCCA repeat in the TK2 transcript in SCA31 when the repeat unit is shifted by one base.

[0006] However, no effective treatments for the above-mentioned SCA31 and SCA10 have been known to date.

[0007] Nagaoka U., Takashima M., Ishikawa K., et al., Neurology, 2000, 54:1971-1975.Sato N., Amino T., Kobayashi K., et al., Am J Hum Genet, 2009, 85:544-557.McFarland KN, et al., PLoS One, 2015, 10(8):e0135906.

[0008] The object of the present invention is to provide a nucleic acid molecule that has an antisense effect on the transcript of a mutant gene containing an abnormal repeat region.

[0009] To achieve the above object, the present inventors conducted extensive research to identify nucleic acid molecules that have antisense effects on the transcripts of mutant genes containing SCA31 abnormal repeat regions. As a result, they discovered a group of nucleic acid molecules that can effectively suppress the expression of the transcripts. The present invention is based on the above findings and provides the following:

[0010] (1) A nucleic acid molecule having an antisense effect against a transcript of a mutant gene containing an abnormal repeat region, wherein the abnormal repeat region consists of a 5' region, a core repeat region, and a 3' region, wherein the core repeat region consists of a base sequence with multiple repeats of the base sequence shown in SEQ ID NO: 1 (5'-TGGAA-3') or a base sequence complementary thereto, and the nucleic acid molecule contains a base sequence complementary to four or more consecutive bases in the abnormal repeat region in the transcript and a target binding region capable of hybridizing to the transcript. (2) The nucleic acid molecule of (1), wherein the mutant gene is a mutant NEDD4-associated brain expression 1 (BEAN1) gene, wherein the core repeat region consists of a base sequence with multiple repeats of the base sequence shown in SEQ ID NO: 1 (5'-TGGAA-3'), and the 5' region contains the base sequence shown in SEQ ID NO: 2 (5'-TCAC-3'). (3) The nucleic acid molecule according to (2), wherein the 5' region consists of any one of the base sequences selected from the group consisting of (a1) to (a4) below: (a1) the base sequence shown in SEQ ID NO: 3; (a2) the base sequence shown in SEQ ID NO: 4; (a3) ​​the base sequence shown in SEQ ID NO: 2 (5'-TCAC-3'); and (a4) a base sequence formed by linking, in order from the 5' end, the base sequence shown in SEQ ID NO: 2 (5'-TCAC-3') and the base sequence shown in SEQ ID NO: 5 (5'-TAGAA-3'). (4) A nucleic acid molecule according to any of (1) to (3), wherein the mutant gene is a mutant NEDD4-associated brain expression 1 (BEAN1) gene, the core repeat region consists of a base sequence in which the base sequence shown in SEQ ID NO: 1 (5'-TGGAA-3') is repeated multiple times, and the 3' region includes a sequence in which the base sequence shown in SEQ ID NO: 5 (5'-TAGAA-3') is repeated multiple times and a sequence in which the base sequence shown in SEQ ID NO: 6 is repeated multiple times. (5) The nucleic acid molecule according to (4), wherein the 3' region consists of any one of the base sequences selected from the group consisting of (b1) to (b5) below:(b1) a base sequence obtained by linking, in order from the 5' end, the base sequence shown in SEQ ID NO: 7, the base sequence obtained by repeating the base sequence shown in SEQ ID NO: 5 (5'-TAGAA-3') multiple times, and the base sequence obtained by repeating the base sequence shown in SEQ ID NO: 6 multiple times; (b2) a base sequence obtained by linking, in order from the 5' end, the base sequence shown in SEQ ID NO: 9, the base sequence obtained by repeating the base sequence shown in SEQ ID NO: 5 (5'-TAGAA-3') multiple times, and the base sequence obtained by repeating the base sequence shown in SEQ ID NO: 6 multiple times; (b3) a base sequence obtained by linking, in order from the 5' end, the base sequence shown in SEQ ID NO: 11, the base sequence obtained by repeating the base sequence shown in SEQ ID NO: 5 (5'-TAGAA-3') multiple times, and the base sequence obtained by repeating the base sequence shown in SEQ ID NO: 6 multiple times; (b4) A base sequence in which the nucleotide sequence shown in SEQ ID NO: 12, the nucleotide sequence shown in SEQ ID NO: 5 (5'-TAGAA-3') is repeated multiple times, the nucleotide sequence shown in SEQ ID NO: 6 is repeated multiple times, and the nucleotide sequence shown in SEQ ID NO: 13 are linked in order from the 5' end, and (b5) A base sequence in which the nucleotide sequence shown in SEQ ID NO: 14, the nucleotide sequence shown in SEQ ID NO: 5 (5'-TAGAA-3') is repeated multiple times, and the nucleotide sequence shown in SEQ ID NO: 6 is repeated multiple times, and the nucleotide sequence is linked in order from the 5' end. (6) The nucleic acid molecule according to any of (2) to (5), wherein the target binding region comprises a non-mutation region adjacent to the 5' side of the 5' region, the 5' region, the core repeat region, the 3' region, and bases complementary to the terminal bases of two adjacent regions in the non-mutation region adjacent to the 3' side of the 3' region. (7) The nucleic acid molecule according to any one of (2) to (5), wherein the entire length of the target binding region is comprised in a nucleotide sequence complementary to either the core repeat region or the 3' region. (8) The nucleic acid molecule according to any one of (2) to (5), wherein the entire length of the target binding region is comprised in a nucleotide sequence complementary to the core repeat region. (9) The nucleic acid molecule according to any one of (2) to (5), consisting of any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 156 to 189 and 207 to 209 (with the proviso that u in the sequences may be t, and t may be u).(10) The nucleic acid molecule according to (1), wherein the mutant gene is a mutant thymidine kinase 2 (TK2) gene, the core repeat region consists of a nucleotide sequence complementary to a nucleotide sequence comprising multiple repeats of the nucleotide sequence shown in SEQ ID NO: 1 (5'-TGGAA-3'), and the 5' region comprises a sequence comprising multiple repeats of the nucleotide sequence shown in SEQ ID NO: 16 and a sequence comprising multiple repeats of the nucleotide sequence shown in SEQ ID NO: 17 (5'-TTCTA-3'). (11) The nucleic acid molecule according to (10), wherein the 5' region consists of any nucleotide sequence selected from the group consisting of the following (c1) to (c5): (c1) a base sequence in which the base sequence shown in SEQ ID NO: 16 is repeated multiple times, a base sequence in which the base sequence shown in SEQ ID NO: 17 (5'-TTCTA-3') is repeated multiple times, and a base sequence in which the base sequence shown in SEQ ID NO: 19 is linked in order from the 5' end; (c2) a base sequence in which the base sequence shown in SEQ ID NO: 16 is repeated multiple times, a base sequence in which the base sequence shown in SEQ ID NO: 17 (5'-TTCTA-3') is repeated multiple times, and a base sequence in which the base sequence shown in SEQ ID NO: 21 is linked in order from the 5' end; (c3) a base sequence in which the base sequence shown in SEQ ID NO: 16 is repeated multiple times, a base sequence in which the base sequence shown in SEQ ID NO: 17 (5'-TTCTA-3') is repeated multiple times, and a base sequence in which the base sequence shown in SEQ ID NO: 22 is linked in order from the 5' end; (c4) A nucleic acid molecule according to any one of (1), (10), and (11), wherein the mutant gene is a mutant thymidine kinase 2 (TK2) gene, the core repeat region is a base sequence complementary to a base sequence comprising multiple repeats of the base sequence of SEQ ID NO: 1 (5'-TGGAA-3'), and the 3' region comprises the base sequence of SEQ ID NO: 26 (5'-GTGA-3').(13) The nucleic acid molecule according to (12), wherein the 3' region consists of any one of the nucleotide sequences selected from the group consisting of (d1) to (d4) below: (d1) the nucleotide sequence shown in SEQ ID NO: 27, (d2) the nucleotide sequence shown in SEQ ID NO: 28, (d3) the nucleotide sequence shown in SEQ ID NO: 26 (5'-GTGA-3'), and (d4) a nucleotide sequence formed by linking, in order from the 5' end, a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 17 (5'-TTCTA-3') is repeated multiple times with the nucleotide sequence shown in SEQ ID NO: 26 (5'-GTGA-3'). (14) The nucleic acid molecule according to any of (10) to (13), wherein the target binding region comprises each of bases complementary to the terminal bases of the 5' region, the core repeat region, the 3' region, and two adjacent regions in the non-mutation region adjacent to the 3' side of the 3' region. (15) The nucleic acid molecule according to any one of (10) to (13), wherein the entire length of the target binding region is comprised in a base sequence complementary to either the 5' region or the core repeat region. (16) The nucleic acid molecule according to any one of (10) to (13), wherein the entire length of the target binding region is comprised in a base sequence complementary to the core repeat region. (17) The nucleic acid molecule according to any one of (10) to (13), consisting of any one base sequence selected from the group consisting of SEQ ID NOs: 33 to 53, 58 to 139, 141 to 150, 152 to 155, and 204 to 206 (with the proviso that u in the sequences may be t, and t may be u). (18) The nucleic acid molecule according to (1), wherein the mutant gene is a mutant ataxin 10 (ATXN10) gene, the core repeat region consists of a nucleotide sequence complementary to a nucleotide sequence in which the nucleotide sequence (5'-TGGAA-3') shown in SEQ ID NO: 1 is repeated multiple times, and the entire length of the target binding region is contained in the nucleotide sequence complementary to the core repeat region. (19) The nucleic acid molecule according to (18), wherein the nucleic acid molecule consists of any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 33 to 37, 58 to 61, 64 to 67, 70 to 139, 141 to 150, 152 to 155, and 204 to 206 (with the proviso that u in the sequence may be t, and t may be u). (20) The nucleic acid molecule according to any of (1) to (19), which is 12 to 30 bases in length. (21) The nucleic acid molecule according to any of (1) to (20), which is a mixmer. (22) The nucleic acid molecule according to any one of (1) to (20), which is a gapmer.(23) The nucleic acid molecule according to (22), comprising: [1] a central region comprising at least two consecutive deoxyribonucleosides; [2] a 5'-wing region comprising an unnatural nucleoside located on the 5'-end of the central region; and [3] a 3'-wing region comprising an unnatural nucleoside located on the 3'-end of the central region. (24) The nucleic acid molecule according to (23), wherein the 5'-wing region and the 3'-wing region comprise a bridged nucleoside and / or a 2'-modified nucleoside. (25) The nucleic acid molecule according to (24), wherein the bridged nucleoside is an LNA nucleoside or an ENA nucleoside. (26) The nucleic acid molecule according to (24) or (25), wherein the 2'-modified group of the 2'-modified nucleoside is a 2'-O-methyl group or a 2'-O-methoxyethyl group. (27) The nucleic acid molecule according to any one of (1) to (20), comprising or consisting of a morpholino nucleic acid. (28) The nucleic acid molecule according to any one of (1) to (27), wherein all or part of the internucleoside linkages of the nucleic acid molecule are modified internucleoside linkages. (29) The nucleic acid molecule according to (28), wherein the modified internucleoside linkages are phosphorothioate linkages. (30) The nucleic acid molecule according to any one of (1) to (29), comprising a modified nucleobase. (31) The nucleic acid molecule according to any one of (1) to (30), wherein the antisense effect is a decrease in the amount of the transcript (preferably a decrease due to degradation of the transcript). (32) The nucleic acid molecule according to any one of (1) to (30), wherein the antisense effect is a steric block.

[0011] (33) A double-stranded nucleic acid complex comprising a first nucleic acid strand consisting of the nucleic acid molecule according to any one of (1) to (32) and a second nucleic acid strand comprising a base sequence complementary to the first nucleic acid strand. (34) The double-stranded nucleic acid complex according to (33), wherein the second nucleic acid strand comprises ribonucleosides, deoxyribonucleosides, and / or modified nucleosides. (35) The double-stranded nucleic acid complex according to (33) or (34), wherein all nucleosides in the region of the second nucleic acid strand consisting of a base sequence complementary to the central region of the first nucleic acid strand are (a) deoxyribonucleosides, (b) deoxyribonucleosides and ribonucleosides, (c) deoxyribonucleosides and 2'-modified nucleosides, (d) ribonucleosides and 2'-modified nucleosides, or (e) deoxyribonucleosides, ribonucleosides, and 2'-modified nucleosides. (36) A double-stranded nucleic acid complex comprising a first nucleic acid strand consisting of the nucleic acid molecule according to any one of (23) to (26) and a second nucleic acid strand comprising a base sequence complementary to the first nucleic acid strand, wherein the second nucleic acid strand comprises a region comprising at least two consecutive ribonucleosides and / or deoxyribonucleosides complementary to at least two consecutive deoxyribonucleosides in the central region of the first nucleic acid strand. (37) The double-stranded nucleic acid complex according to (36), wherein the second nucleic acid strand comprises a modified internucleoside bond in a region comprising a base sequence complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand. (38) The double-stranded nucleic acid complex according to (37), wherein the modified internucleoside bond is a phosphorothioate bond. (39) The double-stranded nucleic acid complex according to any one of (36) to (38), wherein the second nucleic acid strand contains a bridged nucleoside and / or a 2'-modified nucleoside in a region consisting of a base sequence complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand. (40) In the second nucleic acid strand, the bridged nucleoside is an LNA nucleoside, an ENA nucleoside, or a BNA nucleoside. NC(41) The double-stranded nucleic acid complex according to (39), wherein the 2'-modified group of the 2'-modified nucleoside in the second nucleic acid strand is a 2'-O-methyl group or a 2'-O-methoxyethyl group. (42) The double-stranded nucleic acid complex according to any one of (33) to (41), wherein the second nucleic acid strand comprises one or more 2'-O-methoxyethyl-modified nucleosides. (43) The double-stranded nucleic acid complex according to (42), wherein at least 20% of the total number of nucleosides in the second nucleic acid strand are 2'-O-methoxyethyl-modified nucleosides. (44) The double-stranded nucleic acid complex according to any one of (33) to (43), wherein the second nucleic acid strand comprises one or two or more consecutive 2'-O-methoxyethyl-modified nucleosides located at the 5'-terminus and / or one or two or more consecutive 2'-O-methoxyethyl-modified nucleosides located at the 3'-terminus. (45) The double-stranded nucleic acid complex according to any one of (33) to (44), wherein the second nucleic acid strand comprises 1 to 7 2'-O-methoxyethyl-modified nucleosides at a position other than the 5'-terminus and the 3'-terminus. (46) The double-stranded nucleic acid complex according to any one of (33) to (45), wherein all nucleosides in the second nucleic acid strand other than 2'-O-methoxyethyl-modified nucleosides are deoxyribonucleosides. (47) The double-stranded nucleic acid complex according to any one of (33) to (45), wherein all of the nucleosides in the second nucleic acid strand are 2'-O-methoxyethyl-modified nucleosides. (48) The double-stranded nucleic acid complex according to any one of (33) to (47), wherein the second nucleic acid strand comprises a modified nucleic acid base. (49) The double-stranded nucleic acid complex according to any one of (33) to (48), wherein the second nucleic acid strand is bound to tocopherol, cholesterol, or an analog thereof.

[0012] (50) The double-stranded nucleic acid complex according to any one of claims 33 to 49, wherein the base sequence of the first nucleic acid strand consists of the base sequence set forth in SEQ ID NO: 65 (provided that t in the sequence may be u), and the base sequence of the second nucleic acid strand consists of any base sequence selected from the group consisting of SEQ ID NO: 140, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, and SEQ ID NO: 203 (provided that t in the sequence may be u). (51) The double-stranded nucleic acid complex according to claim 50, wherein the first nucleic acid strand consists of the nucleic acid strand set forth in SEQ ID NO: 65, and the second nucleic acid strand consists of any nucleic acid strand selected from the group consisting of SEQ ID NO: 140, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, and SEQ ID NO: 203. (52) A pharmaceutical composition for treating spinocerebellar ataxia type 31 (SCA31), comprising the nucleic acid molecule according to any one of (2) to (9) and (20) to (32) citing them, or the double-stranded nucleic acid complex according to any one of (33) to (49) citing them, and / or the nucleic acid molecule according to any one of (10) to (19) and (20) to (32) citing them, or the double-stranded nucleic acid complex according to any one of (33) to (51) citing them. (53) A pharmaceutical composition for treating spinocerebellar ataxia type 10 (SCA10), comprising the nucleic acid molecule according to any one of (18) or (19) and (20) to (32) citing them, or the double-stranded nucleic acid complex according to any one of (33) to (51) citing them. (54) The pharmaceutical composition according to (52) or (53), which is administered intracerebroventricularly or intrathecally. (55) The pharmaceutical composition according to any one of (52) to (54), wherein the nucleic acid molecule is administered in a single dose of 0.1 mg / kg or more. (56) A method for treating spinocerebellar ataxia type 31 (SCA31), comprising administering to a patient an effective amount of the nucleic acid molecule according to any one of (2) to (9) and (20) to (32) citing therein, or the double-stranded nucleic acid complex according to any one of (33) to (49) citing therein, and / or the nucleic acid molecule according to any one of (10) to (19) and (20) to (32) citing therein, or the double-stranded nucleic acid complex according to any one of (33) to (51) citing therein.(57) A method for treating spinocerebellar ataxia type 10 (SCA10), comprising administering to a patient an effective amount of the nucleic acid molecule according to any one of (18) or (19) and (20) to (32) citing (18) or (19), or the double-stranded nucleic acid complex according to any one of (33) to (51) citing (18) or (19). (58) A nucleic acid molecule according to any one of (2) to (9) and (20) to (32) citing (18) or the double-stranded nucleic acid complex according to any one of (33) to (49) citing (18) or (19), or the double-stranded nucleic acid complex according to any one of (33) to (51) citing (18) or (19), for use in treating spinocerebellar ataxia type 31 (SCA31). (59) The nucleic acid molecule according to any one of (18) or (19) and (20) to (32) citing (18) or (19), or the double-stranded nucleic acid complex according to any one of (33) to (51) citing (18) or (19), for use in the treatment of spinocerebellar ataxia type 10 (SCA10). (60) Use of the nucleic acid molecule according to any one of (2) to (9) and (20) to (32) citing (18) or the double-stranded nucleic acid complex according to any one of (33) to (49) citing (18) or (19), for the manufacture of a therapeutic agent for spinocerebellar ataxia type 31 (SCA31). (61) Use of the nucleic acid molecule according to any one of (18) or (19) and (20) to (32) citing (18) or (19), or the double-stranded nucleic acid complex according to any one of (33) to (51) citing (18) or (19), for the manufacture of a therapeutic agent for spinocerebellar ataxia type 10 (SCA10). This specification includes the disclosures of Japanese Patent Application No. 2021-181571, from which the present application claims priority.

[0013] Nucleic acid molecules can be provided that have an antisense effect on the transcript of a mutant gene that contains an abnormal repeat region.

[0014] FIG. 1 shows the structures of various natural and non-natural nucleotides. FIG. 2 shows the structures of various bridged nucleic acids. FIG. 3 is a schematic diagram showing an example of a specific embodiment of a nucleic acid molecule used in the present invention. FIG. 4 is a schematic diagram showing an example of a specific embodiment of a nucleic acid complex used in the present invention. FIG. 5 shows the structure of a mutant TK2 gene containing an abnormal repeat region. FIG. 5A shows the location of the abnormal repeat region in the mutant TK2 gene. The non-mutated sequence adjacent to the SCA31 abnormal repeat region contains a polymorphic sequence (5'-(TAAAA) m-3'; m is an integer of 1 or more, e.g., approximately 8 to 20). Figure 5B shows known sequences for the 5' region, core repeat region, and 3' region. Figure 6 is a schematic diagram showing the structure of the antisense oligonucleotides (ASOs) used in Example 1 and their location in the mutant TK2 gene. In the designation of each ASO, capital letters indicate DNA, and underlined capital letters indicate LNA (underlined C indicates 5-methylcytosine LNA). Figure 7 shows the inhibitory effect of ASO (2 nM) on the expression of SCA31 mutant repeat (TK2) in Example 2. Error bars indicate standard error. Figure 8 shows the results of FISH to detect the expression of SCA31 mutant repeat (TK2) in Example 2. T-r1 (2 nM) was used as the ASO against SCA31 mutant repeat (TK2). Cells in which expression of SCA31 mutant repeat (TK2) was detected are indicated by arrows. Figure 9 shows the inhibitory effect of ASO (0.1 nM, 0.5 nM, or 2 nM) on the expression of the SCA31 mutant repeat (TK2) in Example 4. Error bars indicate standard error. Figure 10 shows the inhibitory effect of ASO (0.1 nM, 0.5 nM, 1 nM, or 2 nM) on the expression of the SCA31 mutant repeat (TK2) in Example 5. Error bars indicate standard error. Figure 11 shows the inhibitory effect of ASO (0.1 nM) on the expression of the SCA31 mutant repeat (TK2) in Example 6. Error bars indicate standard error. Figure 12 shows the structure of an ASO targeting the core repeat region. In the representation of each ASO, uppercase letters indicate DNA, underlined uppercase letters indicate ENA, and italicized lowercase letters indicate 2'-OMe RNA. Figure 13 shows the inhibitory effect of ASO (0.1 nM) targeting the core repeat region within SCA31 mutant repeat (TK2) expression in Example 7. Error bars indicate standard error. Figure 14 shows the structure of ASO targeting the core repeat region within SCA31 mutant repeat (TK2). In the representation of each ASO, capital letters indicate DNA, underlined capital letters indicate LNA, and italicized lowercase letters indicate 2'-O-MOE RNA. Figure 15 shows the inhibitory effect of ASO (0.5 nM or 2 nM) targeting the core repeat region within SCA31 mutant repeat (TK2) expression in Example 8.Figure 16 is a graph showing the inhibitory effect of ASO (0.5 nM) on the expression of the SCA31 mutation repeat (TK2) in Example 9. Error bars indicate standard error. Figure 17 is a graph showing the inhibitory effect of ASO (0.5 nM) on the expression of the SCA31 mutation repeat (TK2) in Example 10. Error bars indicate standard error. Figure 18 is a graph showing the inhibitory effect of ASO (0.5 nM) on the expression of the SCA31 mutation repeat (TK2) in Example 11. Error bars indicate standard error. Figure 19 is a graph showing the inhibitory effect of ASO (0.5 nM) on the expression of the SCA31 mutation repeat (TK2) in Example 12. Error bars indicate standard error. Figure 20 is a graph showing the inhibitory effect of heteroduplex nucleic acid (0.5 nM) on the expression of the SCA31 mutation repeat (TK2) in Example 14. Error bars indicate standard error. Figure 21 shows the inhibitory effect of ASO on the expression of the SCA31 mutant repeat (TK2) in Example 15. LNA / DNA gapmers (LDG), LNA / RNA gapmers (LRG), LNA / DNA mixmers (LDM), and LNA / RNA mixmers (LRM) were used as ASOs. Error bars indicate standard error. Figure 22 shows the structure of a mutant BEAN1 gene containing an abnormal repeat region. Figure 22A shows the location of the abnormal repeat region in the mutant BEAN1 gene. The non-mutated sequence adjacent to the SCA31 abnormal repeat region contains a polymorphic sequence (5'-(TAAAA)). m-3'; m is an integer of 1 or more, e.g., about 8 to 20). Figure 22B shows known sequences for the 5' region, core repeat region, and 3' region. Figure 23 is a schematic diagram showing the structure of the ASO used in Example 16 and its location in the mutant BEAN1 gene. In the representation of each ASO, capital letters indicate DNA, and underlined capital letters indicate LNA (the underlined C indicates 5-methylcytosine LNA). Figure 24 shows the effect of ASO (2 nM) in Example 16 on the suppression of expression of the SCA31 mutant repeat (BEAN1). Error bars indicate standard error. Figure 25 shows the effect of ASO (0.1 nM) in Example 18 on the suppression of expression of the SCA31 mutant repeat (BEAN1). Error bars indicate standard error. Figure 26 shows the structure of an ASO targeting the core repeat region. In the representation of each ASO, capital letters indicate DNA, and italicized lowercase letters indicate 2'-O-MOE RNA. Figure 27 shows the inhibitory effect of ASO (0.1 nM) targeting the core repeat region within SCA31 (BEAN1) in Example 19. Figure 28 shows the inhibitory effect of ASO on SCA31 (BEAN1) in Example 20. LNA / DNA gapmers (LDG), LNA / RNA gapmers (LRG), LNA / DNA mixmers (LDM), and LNA / RNA mixmers (LRM) were used as ASOs. Error bars indicate standard error. Figure 29 shows the structures of the transgene constructs used to generate TK2-Tg and BEAN1-BAC-Tg mice. "SCA31-1" and "EX2-3" indicate the locations detected by qRT-PCR. Figure 30 shows the injection locations into mouse ventricles in Example 21. "+" indicates Bregma, and "○" indicates the injection location. Figure 31 shows the effect of ASO or HDO on the suppression of SCA31 mutation repeat (TK2) expression in TK2-Tg mice in Example 21. Figure 31A shows the results of qRT-PCR demonstrating the effect of T-re10a ASO on the suppression of SCA31 mutation repeat (TK2) expression. Tests were performed using TK2-Tg mice, and the average values ​​are shown. Figure 31B shows the results of qRT-PCR demonstrating the effect of T-re10a-HDO on the suppression of SCA31 mutation repeat (TK2) expression.Error bars indicate standard error. Figure 31C is a Northern blot result showing the suppressive effect of T-re10a-HDO on the expression of the SCA31 mutant repeat (TK2). The position of the band corresponding to the SCA31 mutant repeat (TK2) is indicated by an arrow. Figure 32 is a diagram showing the suppressive effect of ASO on the expression of the SCA31 mutant repeat in Example 21. Figure 32A is a qRT-PCR result showing the suppressive effect of T-r2'_1 ASO on the expression of the SCA31 mutant repeat (TK2) in TK2-Tg mice. Figure 32B is a qRT-PCR result showing the suppressive effect of B-r2 ASO on the expression of the SCA31 mutant repeat (BEAN1) in BEAN-BAC-Tg mice. Error bars indicate standard error. Figure 33 is a schematic diagram showing the structure of the HDO used in Example 22. Figure 33A shows the structure of T-re10a HDO (Default). Figure 33B shows the structure of T-re10a HDO (cMOE / DNA). Figure 33C shows the structure of T-re10a HDO (Full cMOE). Figure 33D shows the structure of T-re10a HDO (cMOE / DNA_2). Figure 33E shows the structure of T-re10a HDO (cMOE / DNA_3). Figure 34 shows the effect of HDO on the suppression of expression of the SCA31 mutant repeat (TK2) in Example 22. Error bars indicate standard error. Figure 35 shows the effect of HDO on the suppression of expression of the SCA31 mutant repeat (TK2) in Example 22. Error bars indicate standard error. Figure 36 shows the structure of a pkSCX-IRES-EGFP vector containing an SCA31 insertion sequence in the TK2 or BEAN1 orientation. Figure 36A shows a pkSCX-IRES-EGFP vector containing an SCA31 insertion sequence in the TK2 orientation. Figure 36B shows the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the BEAN1 direction. "SCA31-0" indicates the position detected by qRT-PCR. Figure 37 shows the effect of HDO in suppressing the expression of the SCA31 mutant repeat (TK2) in Example 22. The figure shows the average results (n = 3 to 9) in the left cerebellum, and the error bars indicate the standard error.

[0015] <Nucleic Acid Molecules> In one aspect, the present invention relates to nucleic acid molecules. The nucleic acid molecules of the present invention have an antisense effect on transcripts of mutant genes containing abnormal repeat regions. The nucleic acid molecules of the present invention contain a target binding region that contains a base sequence complementary to four or more consecutive bases in the abnormal repeat region and is capable of hybridizing to transcripts of mutant genes containing the abnormal repeat region.

[0016] As used herein, the term "abnormal repeat region" refers to a region consisting of an abnormal nucleotide sequence found in a mutant gene (mutated BEAN1 gene, mutant TK2 gene, or mutant ATXN10 gene) in a patient with spinocerebellar ataxia type 31 (SCA31) or type 10 (SCA10). The term "abnormal repeat region" refers to the entire region of abnormal sequence that is not present in the gene of a normal individual and is present only in SCA31 or SCA10, and is used herein as a term that can encompass both repetitive and non-repetitive sequence portions. The abnormal repeat region is either a region consisting of an abnormal nucleotide sequence contained in an intron of a mutant BEAN1 gene in a patient with spinocerebellar ataxia type 31 (SCA31), a region consisting of an abnormal nucleotide sequence contained in an intron of a mutant TK2 gene in a patient with spinocerebellar ataxia type 31 (SCA31), or a region consisting of an abnormal nucleotide sequence contained in an intron of a mutant ATXN10 gene in a patient with allele B of spinocerebellar ataxia type 10 (SCA10). In this specification, the abnormal repeat region in SCA31 and SCA10 is referred to as the "SCA31 abnormal repeat region" and the "SCA10 abnormal repeat region," respectively. The specific location of the SCA31 abnormal repeat region can be identified, for example, as between positions 68486 and 68487 in the NCBI Reference Sequence: NG_021403.2.

[0017] As used herein, the term "target gene" refers to a gene to which the nucleic acid molecule of the present invention or the first nucleic acid strand of a double-stranded nucleic acid complex can bind. Specifically, the target gene refers to any of a mutant BEAN1 gene containing an abnormal repeat region, a mutant TK2 gene containing an abnormal repeat region, and a mutant ATXN10 gene containing an abnormal repeat region.

[0018] As used herein, the term "target transcript" refers to an RNA that is directly targeted by the nucleic acid molecule or nucleic acid complex of the present invention and is synthesized by RNA polymerase. Specifically, it refers to an RNA that is transcribed from a mutant gene targeted by the present invention and contains an RNA sequence corresponding to an abnormal repeat region. It may be a mature mRNA, a pre-mRNA, or an RNA fragment derived from a pre-mRNA (e.g., an intron portion spliced ​​out from a pre-mRNA), but is preferably a pre-mRNA. Examples of target transcripts include RNA that is a transcription product of a mutant BEAN1 gene, RNA that is a transcription product of a mutant TK2 gene, and RNA that is a transcription product of a mutant ATXN10 gene.

[0019] As used herein, the term "antisense oligonucleotide (ASO)" or "antisense nucleic acid" refers to a single-stranded oligonucleotide that contains a complementary base sequence capable of hybridizing to all or a part of a target transcript, for example, any target region, and that can control the expression of the transcript of the target gene or the level of the target transcript through its antisense effect.

[0020] As used herein, the term "antisense effect" refers to the effect of ASOs hybridizing to a target transcript to modulate its expression or editing. "Modulating the expression or editing of a target transcript" refers to suppression or reduction of target gene expression or target transcript expression levels (herein, "target transcript expression levels" is often referred to as "target transcript levels"), translation inhibition, RNA editing, splicing function alteration effects (e.g., splicing switches, exon inclusion, exon skipping, etc.), or transcript degradation. For example, in post-transcriptional inhibition of a target gene, when an RNA oligonucleotide is introduced into a cell as an ASO, the ASO forms a partial duplex with the mRNA, the transcript of the target gene. This partial duplex acts as a cover to prevent ribosomal translation, thereby inhibiting the expression of the target protein encoded by the target gene at the translational level (steric blocking). On the other hand, when an oligonucleotide containing DNA is introduced into a cell as an ASO, a partial DNA-RNA heteroduplex is formed. This heteroduplex structure is recognized by RNase H, resulting in degradation of the target gene's mRNA and inhibition of the expression of the protein encoded by the target gene. Furthermore, antisense effects can also be achieved by targeting introns in pre-mRNA. Furthermore, antisense effects can also be achieved by targeting non-coding RNAs such as miRNAs (steric blocking). In this case, inhibition of the function of the non-coding RNA can affect the expression and function of the gene targeted by the non-coding RNA. For example, inhibition of miRNA function can increase the expression of genes whose expression is normally controlled by the miRNA. In one embodiment, the antisense effect is a reduction in transcript abundance and / or steric blocking. Generally, when an ASO containing DNA is introduced into a cell, the ASO hybridizes to a target gene mRNA with a sequence complementary to the ASO, forming a DNA-RNA heteroduplex. RNase H recognizes this heteroduplex structure and degrades the target gene mRNA.Therefore, the reduction in the amount of the transcript is preferably brought about by degradation of the transcript.

[0021] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to a monomer, a nucleoside or nucleotide, an oligomer, an oligonucleotide, or a polymer, a polynucleotide.

[0022] "Nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar portion of a nucleoside is typically, but not limited to, a pentofuranosyl sugar, specific examples of which include ribose and deoxyribose. The base portion (nucleobase) of a nucleoside is typically a heterocyclic base moiety. Examples include, but are not limited to, adenine, cytosine, guanine, thymine, or uracil, as well as other modified nucleobases (modified bases).

[0023] A "nucleotide" refers to a molecule in which a phosphate group is covalently linked to the sugar portion of a nucleoside. In the case of nucleotides containing a pentofuranosyl sugar, the phosphate group is typically linked to the 2', 3', or 5' hydroxyl group of the sugar.

[0024] An "oligonucleotide" refers to a linear oligomer formed by covalently linking several to several dozen hydroxyl groups and phosphate groups in the sugar moieties of adjacent nucleotides. A "polynucleotide" refers to a linear polymer formed by linking several dozens, preferably several hundred, of nucleotides, more numerous than an oligonucleotide, by such covalent bonds. The nucleosides of an oligonucleotide or polynucleotide are linked by phosphodiester bonds.

[0025] As used herein, the term "nucleic acid strand" or simply "strand" refers to an oligonucleotide or polynucleotide. A nucleic acid strand can be produced in full length or in partial form by chemical synthesis, for example, using an automated synthesizer, or by enzymatic processes using polymerases, ligases, or restriction enzyme reactions. A nucleic acid strand can contain natural and / or non-natural nucleotides.

[0026] As used herein, "natural nucleosides" refer to nucleosides that exist in nature. Examples include ribonucleosides consisting of ribose and a base such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides consisting of deoxyribose and a base such as adenine, cytosine, guanine, or thymine. Ribonucleosides found in RNA and deoxyribonucleosides found in DNA are often referred to herein as "DNA nucleosides" and "RNA nucleosides," respectively.

[0027] As used herein, the term "natural nucleotide" refers to a naturally occurring nucleotide molecule in which a phosphate group is covalently bound to the sugar moiety of the natural nucleoside. Examples include ribonucleotides, which are known as building blocks of RNA and in which a phosphate group is bound to a ribonucleoside, and deoxyribonucleotides, which are known as building blocks of DNA and in which a phosphate group is bound to a deoxyribonucleoside.

[0028] As used herein, the term "unnatural nucleoside" refers to any nucleoside other than a natural nucleoside. For example, this term includes modified nucleosides and nucleoside mimetics. As used herein, the term "modified nucleoside" refers to a nucleoside having a modified sugar moiety and / or a modified nucleobase. Nucleic acid chains, including unnatural oligonucleotides, are often preferred over natural forms due to desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0029] As used herein, the term "mimetic" refers to functional groups that replace sugars, nucleobases, and / or internucleoside linkages. Generally, mimetics are used in place of sugars or sugar-internucleoside linkage combinations, while maintaining the nucleobases for hybridization to a selected target. The term "nucleoside mimetic" includes structures used to replace sugars, sugars and bases, or linkages between monomeric subunits at one or more positions in an oligomeric compound. An "oligomeric compound" refers to a polymer of linked monomeric subunits that is capable of hybridizing at least to a region of a nucleic acid molecule. Nucleoside mimetics include, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic, or tricyclic sugar mimetics, e.g., nucleoside mimetics with non-furanose sugar units. Figure 1 shows the structures of various natural and unnatural nucleotides.

[0030] As used herein, the term "bicyclic nucleoside" refers to a modified nucleoside containing a bicyclic sugar moiety. Nucleic acids containing a bicyclic sugar moiety are commonly referred to as bridged nucleic acids (BNAs). As used herein, nucleosides containing a bicyclic sugar moiety may also be referred to as "bridged nucleosides." Some examples of bridged nucleic acids are shown in Figure 2.

[0031] A bicyclic sugar may be a sugar in which the 2' and 4' carbon atoms are bridged by two or more atoms. Examples of bicyclic sugars are known to those skilled in the art. One subgroup of nucleic acids (BNAs) containing bicyclic sugars is the 4'-(CH2) p -O-2',4'-(CH2) p -CH2-2',4'-(CH2) p -S-2',4'-(CH2) p -OCO-2',4'-(CH2) n -N(R3)-O-(CH2) m-2' [wherein p, m, and n represent an integer of 1 to 4, an integer of 0 to 2, and an integer of 1 to 3, respectively; and R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, or a unit substituent (a fluorescent or chemiluminescent labeled molecule, a functional group having nucleic acid cleavage activity, an intracellular or intranuclear localization signal peptide, etc.)]. Furthermore, with respect to BNAs according to certain embodiments, in the OR2 substituent on the 3' carbon atom and the OR1 substituent on the 5' carbon atom, R1 and R2 are typically hydrogen atoms, but may be the same or different from each other and may also be a protecting group for a hydroxyl group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or P(R4)R5 (wherein R4 and R5 may be the same or different from each other and represent, respectively, a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted with an alkyl group having 1 to 5 carbon atoms).Non-limiting examples of such BNAs include methyleneoxy (4'-CH2-O-2') BNAs (LNAs (Locked Nucleic Acids) Acid®, also known as 2',4'-BNA), for example, α-L-methyleneoxy (4'-CH2-O-2') BNA or β-D-methyleneoxy (4'-CH2-O-2') BNA, ethyleneoxy (4'-(CH2)2-O-2') BNA having an ethylene bridge between the 2- and 4-positions (also known as "2'-O,4'-C-ethylene nucleoside" or "ENA"; see JP 2000-297097 A and WO 2019 / 009299 A1), β-D-thio (4'-CH2-S-2') BNA, aminooxy (4'-CH2-ON(R3)-2') BNA, oxyamino (4'-CH2-N(R3)-O-2') BNA (2',4'-BNA). NC Also known as ; R=H is 2',4'-BNA NC [NH], R=Me is 2',4'-BNA NC [N-Me]), 2',4'-BNA coc , 3'-amino-2',4'-BNA, 5'-methyl BNA, (4'-CH(CH3)-O-2')BNA (also known as cEt BNA), (4'-CH(CHOCH3)-O-2')BNA (also known as cMOE BNA), amide BNA (4'-C(O)-N(R)-2')BNA (R=H, Me) (also known as AmNA; R=H is AmNA[NH], R=Me is AmNA[N-Me])), guanidine BNA (also known as GuNA (e.g., R=H is GuNA[NH], R=Me is GuNA[N-Me] in Figure 5)), amine BNA (also known as 2'-Amino-LNA), 2'-O,4'-C-spirocyclopropylene bridged nucleic acid (also known as scpBNA), and other BNAs known to those skilled in the art. Bicyclic nucleosides with a methyleneoxy (4'-CH2-O-2') bridge are sometimes referred to as LNA nucleosides.

[0032] As used herein, the term "unnatural nucleotide" refers to any nucleotide other than a natural nucleotide, including modified nucleotides and nucleotide mimetics. As used herein, "modified nucleotide" refers to a nucleotide having one or more of a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase. The term "nucleotide mimic" as used herein includes structures used to replace nucleosides and linkages at one or more positions in an oligomeric compound. Examples of nucleotide mimetics include peptide nucleic acids and morpholino nucleic acids. Peptide nucleic acids (PNA) are nucleotide mimetics with a backbone in which N-(2-aminoethyl)glycine is linked via an amide bond instead of a sugar. Morpholino nucleic acids (MNA) are nucleotide mimetics with a backbone in which morpholine rings are linked via phosphorodiamidate linkages. Nucleic acid chains, including unnatural oligonucleotides, often have desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, improved stability in the presence of nucleases, or improved inhibitory activity. Therefore, when nucleotides are utilized as ASOs, non-natural nucleotides are preferred over natural nucleotides.

[0033] As used herein, the term "modified internucleoside linkage" refers to an internucleoside linkage that has a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester linkage). Modified internucleoside linkages include phosphorus-containing internucleoside linkages that contain a phosphorus atom and non-phosphorus-containing internucleoside linkages that do not contain a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphorothioate linkages, phosphorodithioate linkages, phosphotriester linkages, alkylphosphonate linkages, alkylthiophosphonate linkages, and phosphorodiamidates. A phosphorothioate linkage is an internucleoside linkage in which the non-bridging oxygen atom of a phosphodiester bond is replaced with a sulfur atom. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known. The modified internucleoside linkage is preferably one that is more nuclease-resistant than a naturally occurring internucleoside linkage.

[0034] When an internucleoside bond has a chiral center, the internucleoside bond may be chiral controlled. "Chiral controlled" refers to a bond existing as a single diastereomer with respect to the chiral center, e.g., a chiral phosphorus atom. A chiral controlled internucleoside bond may have a completely single stereochemistry or may have a high isomeric purity, e.g., 90% de, 95% de, 98% de, 99% de, 99.5% de, 99.8% de, 99.9% de, or higher. As used herein, "isomeric purity" refers to the proportion of one diastereomer in a mixture of diastereomers, expressed as diastereomeric excess (% de), and defined as (target diastereomer - other diastereomers) / (total diastereomers) x 100 (%).

[0035] For example, the internucleoside linkages may be phosphorothioate linkages that are chiral controlled in the Rp or Sp configuration. Methods for preparing chiral internucleoside bonds are known. For example, phosphorothioate bonds chiral controlled to the Rp or Sp configuration are prepared by the methods described in Naoki Iwamoto et al., Angew. Chem. Int. Ed. Engl. 2009, 48(3), 496-9; Natsuhisa Oka et al., J. Am. Chem. Soc. 2003, 125, 8307-8317; Natsuhisa Oka et al., J. Am. Chem. Soc. 2008, 130, 16031-16037; Yohei Nukaga et al., J. Org. Chem. 2016, 81, 2753-2762; Yohei Nukaga et al., J. Org. Chem. 2012, 77, 7913-7922. Chiral phosphorothioate linkages in the Rp or Sp configuration are also known and are known to produce effects such as those described in Naoki Iwamoto et al., Nat. Biotechnol., 2017, 35(9), 845-851 and Anastasia Khvorova et al., Nat. Biotechnol., 2017, 35(3), 238-248. For example, in one embodiment, phosphorothioate linkages in the Sp configuration are more stable than those in the Rp configuration, and / or ASOs in the Sp configuration are capable of promoting target RNA cleavage by RNase H1, resulting in a more sustained response in vivo.

[0036] As used herein, "modified nucleobase" or "modified base" refers to any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. Examples of modified nucleobases include, but are not limited to, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, N6-methyladenine, 8-bromoadenine, N2-methylguanine, or 8-bromoguanine. A preferred modified nucleobase is 5-methylcytosine. Note that 5-methyluracil and thymine have the same structure, and may be represented as uracil (e.g., "5meU") or thymine (e.g., "T").

[0037] "Unmodified nucleobase" or "unmodified base" is synonymous with natural nucleobases and refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0038] As used herein, the term "modified sugar" refers to a sugar having a substitution and / or any alteration from a natural sugar moiety (i.e., a sugar moiety found in DNA (2'-H) or RNA (2'-OH)). A nucleic acid strand, as used herein, may contain one or more modified nucleosides, optionally including modified sugars. Sugar-modified nucleosides may impart improved nuclease stability, increased binding affinity, or some other beneficial biological property to a nucleic acid strand. A nucleoside may also contain a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), the formation of bridges within the ribofuranose ring to form bicyclic nucleic acids (bridged nucleic acids, BNAs), and the substitution of S, N(R), or C(R1)(R2) of the ribosyl ring oxygen atoms (R, R1, and R2 are each independently H, C1-C 12Examples of nucleosides having modified sugar moieties, as used herein, include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F (2'-fluoro), 2'-OCH (2'-OMe or 2'-O-methyl), and 2'-O(CH)OCH (2'-O-MOE or 2'-O-methoxyethyl).

[0039] As used herein, the term "2'-modified sugar" refers to a furanosyl sugar modified at the 2'-position. 10 Alkyl, aryl, amino, azido, thio, -O-allyl, -O-C1-C 10 alkyl, -OCF3, -O(CH2)2SCH3, -O(CH2)2OCH3, -O(CH2)2-ON(Rm)(Rn), O-CH2-C(=O)-N(Rm)(Rn), and -O-(CH2)2-C(=O)-N(Rm)(Rn), and the like, wherein each Rm and Rn is independently H or a substituted or unsubstituted C1-C 10 As used herein, a nucleoside containing a 2'-modified sugar is referred to as a "2'-modified nucleoside." For example, a 2'-modified nucleoside in which the 2'-modifying group is a 2'-O-methoxyethyl group is referred to as a 2'-O-methoxyethyl-modified nucleoside.

[0040] Generally, modifications can be made so that nucleotides in the same chain can be independently modified. The same nucleotide can also have a modified internucleoside linkage (e.g., a phosphorothioate linkage) and a modified sugar (e.g., a 2'-O-methyl modified sugar or a bicyclic sugar) to confer resistance to enzymatic cleavage. The same nucleotide can also have a modified nucleobase (e.g., a 5-methylcytosine) and a modified sugar (e.g., a 2'-O-methyl modified sugar or a bicyclic sugar).

[0041] The number, type, and position of non-natural nucleotides in a nucleic acid strand can affect the antisense effect, etc., provided by the nucleic acid complex of the present invention. The choice of modification may vary depending on the sequence of the target gene, etc., but those skilled in the art can make an appropriate selection by referring to the descriptions in this specification and literature related to antisense methods (e.g., WO 2007 / 143315, WO 2008 / 043753, and WO 2008 / 049085), and the present invention is not limited to the embodiments described in the Examples. Furthermore, when the antisense effect of a modified nucleic acid complex is measured, if the measured value obtained in this manner is not significantly lower than that of the nucleic acid complex before modification (e.g., if the measured value obtained after modification is 70% or more, 80% or more, or 90% or more of the measured value of the nucleic acid complex before modification), the relevant modification can be evaluated.

[0042] In this specification, A t , G t , 5meC t , C t , T t , U t , A p , G p , 5meC p , C p , T p , U p , A s , G s , 5meC s , C s , T s , U s , A rt , G rt , C rt , U rt , A rp , G rp , C rp , U rp , A rs , G rs , C rs , U rs , A m1t , G m1t , C m1t , 5meC m1t , U m1t , A m1p , G m1p , C m1p , 5meCm1p , U m1p , A m1s , G m1s , C m1s , 5meC m1s , U m1s , A 2t , G 2t , C 2t , T 2t , A e2p , G e2p , C e2p , T e2p , A e2s , G e2s , C e2s , T e2s is a group having the structure shown below.

[0043]

[0044] As used herein, the term "complementary" refers to a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or Wobble base pairs (guanine-thymine or guanine-uracil) through hydrogen bonds. In the present invention, a nucleic acid molecule or a first nucleic acid strand does not necessarily have to be completely complementary to all or a portion of a target transcript (e.g., a transcript of a target gene), but it is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. Similarly, in a double-stranded nucleic acid complex, it is not necessarily required that the second nucleic acid strand be completely complementary to all or a portion of the first nucleic acid strand, but it is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. Sequence complementarity can be determined by using a BLAST program or the like. A first nucleic acid strand can "hybridize" to a target transcript if the sequences are complementary (typically, if the sequence is complementary to at least a portion of the sequence of the target transcript). A first nucleic acid strand can "anneal" to the complementary region of a second nucleic acid strand if the sequences are complementary. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which two strands can anneal or hybridize, taking into account the degree of complementarity between the strands. Such conditions may typically be physiological conditions. Furthermore, those skilled in the art can easily design antisense nucleic acids complementary to a target transcript, for example, based on information on the nucleotide sequence of the target gene.

[0045] Hybridization conditions may be stringent, such as low stringency and high stringency. Low stringency conditions may be, for example, 30°C, 2xSSC, 0.1% SDS. High stringency conditions may be, for example, 65°C, 0.1xSSC, 0.1% SDS. Hybridization stringency can be adjusted by changing conditions such as temperature and salt concentration. Here, 1xSSC contains 150 mM sodium chloride and 15 mM sodium citrate.

[0046] As used herein, "tocopherol" refers to a methylated derivative of tocol, a fat-soluble vitamin (vitamin E) with a ring structure called chroman. Tocol has strong antioxidant properties and therefore functions in vivo as an antioxidant, eliminating free radicals generated by metabolism and protecting cells from damage.

[0047] Several different types of tocopherol are known, consisting of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, based on the position of the methyl group bound to the chroman. The tocopherol referred to herein may be any tocopherol. Examples of tocopherol analogs include various unsaturated analogs of tocopherol, such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol. Preferably, the tocopherol is α-tocopherol.

[0048] As used herein, "cholesterol" refers to a type of sterol, also known as steroid alcohol, which is particularly abundant in animals. Cholesterol plays an important role in metabolic processes in vivo, and is also a major component of the cell membrane system in animal cells, along with phospholipids. Cholesterol analogs refer to various cholesterol metabolites and analogs, which are alcohols having a sterol skeleton, and include, but are not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.

[0049] As used herein, the term "analog" refers to a compound having a similar structure and properties, which has the same or a similar basic skeleton. Analogs include, for example, biosynthetic intermediates, metabolic products, compounds with substituents, etc. Whether a compound is an analog of another compound can be determined by one skilled in the art based on common general technical knowledge.

[0050] As used herein, the term "subject" refers to a target to which the nucleic acid molecule, double-stranded nucleic acid complex, or pharmaceutical composition of the present invention is applied. Subjects include individuals as well as organs, tissues, and cells. When the subject is an individual, it can be any animal, including humans. Non-human subjects include various livestock, poultry, pets, and laboratory animals. The subject may be, but is not limited to, an individual in need of a reduction in the expression level of a target transcript, preferably an individual suffering from or expected to suffer from SCA31 or SCA10 (e.g., an individual belonging to a family with SCA31 or SCA10 and / or an individual expected to suffer from SCA31 or SCA10 based on genotype, etc.).

[0051] In this specification, nucleotide sequences are written in order from the 5' to the 3' end unless otherwise specified. The nucleotide sequences indicated by skipped SEQ ID NOs in the sequence listing are shown below. Note that all of these represent DNA sequences derived from humans. SEQ ID NO: 1: 5'-TGGAA-3' SEQ ID NO: 2: 5'-TCAC-3' SEQ ID NO: 5: 5'-TAGAA-3' SEQ ID NO: 8: 5'-TTCCA-3' SEQ ID NO: 17: 5'-TTCTA-3' SEQ ID NO: 26: 5'-GTGA-3' SEQ ID NO: 191: 5'-TAAAA-3' SEQ ID NO: 194: 5'-TTTTA-3' SEQ ID NO: 196: 5'-TTGAAACAG-3' SEQ ID NO: 198: 5'-CTGTTTCAA-3'

[0052] The nucleic acid molecule of this embodiment contains a target binding region that can hybridize to a transcript of a mutant gene containing an abnormal repeat region and has an antisense effect on the transcript. The target binding region contains a base sequence complementary to four or more consecutive bases in the abnormal repeat region of the transcript.

[0053] The abnormal repeat region contained in the mutant gene encoding the transcript targeted by the nucleic acid molecule of this embodiment consists of a 5' region, a core repeat region, and a 3' region.

[0054] As used herein, the "core repeat region" refers to a nucleotide sequence (5'-(TGGAA) n 8 (5'-TTCCA-3'; n is an integer of 2 or more), or a complementary nucleotide sequence thereto. Here, the "complementary nucleotide sequence thereto" refers to a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 8 (5'-TTCCA-3') is repeated multiple times (5'-(TTCCA) n −3′; n is an integer of 2 or more). Here, “n” varies depending on the case and is not limited to the following range, but examples include integers of 2 to 500, 100 to 400, or 200 to 400.

[0055] As used herein, the "5' region" refers to the region of the abnormal repeat region located 5' to the core repeat region. The "3' region" refers to the region of the abnormal repeat region located 3' to the core repeat region. The nucleotide sequences of the 5' and 3' regions are not limited, as long as they are sequences that can be found in patients with spinocerebellar ataxia SCA31 or SCA10.

[0056] Further configurations of the nucleic acid molecules of the present invention vary depending on the type of mutant gene to be targeted, and will be explained below according to the type of mutant gene.

[0057] In one embodiment, the mutant gene encoding the transcript targeted by the nucleic acid molecule of the present invention is a mutant BEAN1 gene.

[0058] In the abnormal repeat region that may be contained in the mutant BEAN1 gene, the core repeat region is a nucleotide sequence (5'-(TGGAA) n -3'; n is an integer of 2 or more. Here, "n" varies depending on the case and is not limited to the following range, but examples include integers of 2 to 500, 100 to 400, or 200 to 400.

[0059] The nucleotide sequences of the 5' and 3' regions in the abnormal repeat region of the mutant BEAN1 gene are not particularly limited, as long as they are sequences that can be found in patients with spinocerebellar ataxia SCA31.

[0060] The 5' region in the abnormal repeat region of the mutant BEAN1 gene includes, for example, a region containing the nucleotide sequence (5'-TCAC-3') shown in SEQ ID NO: 2. Specific nucleotide sequences include the following (a1) to (a4): (a1) the nucleotide sequence (5'-TCACTAAAA(TAGAA)2-3') shown in SEQ ID NO: 3, (a2) the nucleotide sequence (5'-TCACTAAAA(TAGAA)4-3') shown in SEQ ID NO: 4, (a3) ​​the nucleotide sequence (5'-TCAC-3') shown in SEQ ID NO: 2, and (a4) a nucleotide sequence (5'-(TAGAA) n -3'; n is an integer of 2 or more) linked in order from the 5' side. Here, "n" varies depending on the case and is not limited to the following ranges, but examples include integers of 2 to 500, 100 to 400, or 200 to 400.

[0061] The 3' region of the abnormal repeat region of the mutant BEAN1 gene contains, for example, a sequence in which the nucleotide sequence shown in SEQ ID NO: 5 (5'-TAGAA-3') is repeated multiple times (5'-(TAGAA) n -3'; n is an integer of 2 or more), and a sequence in which the nucleotide sequence shown in SEQ ID NO: 6 is repeated multiple times (5'-(TAAAATAGAA) n Specific examples of such a base sequence include the following (b1) to (b5): (b1) a base sequence (5'-TGGGAATGGAATGGGAA(TAGAA)2(TGGAA)2(TAGAA)2TGGAA-3') shown in SEQ ID NO: 7, a base sequence (5'-(TAGAA) n -3', n is an integer of 2 or more), and a base sequence having the base sequence shown in SEQ ID NO: 6 repeated multiple times (5'-(TAAAATAGAA) n(b2) a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 9 (5'-TGGGAATAGAATGGGAA(TAGAA)2(TGGAA)3TAGAATGGAA-3', n is an integer of 2 or more) is linked in order from the 5' side, n -3', n is an integer of 2 or more), and a base sequence having the base sequence shown in SEQ ID NO: 6 repeated multiple times (5'-(TAAAATAGAA) n (b3) a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 11 (5'-TGGGAATAGAATGGGAA(TGGAA)3(TAGAATGGAA)4(TAGAA)6TGGAATAGAATGGAA-3') and the nucleotide sequence shown in SEQ ID NO: 5 (5'-TAGAA-3') are repeated multiple times (5'-(TAGAA) n -3', n is an integer of 2 or more), and a base sequence having the base sequence shown in SEQ ID NO: 6 repeated multiple times (5'-(TAAAATAGAA) n (b4) a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 12 (5'-(TAGAA)2TGGAA(TAGAA)2TGGAA-3') and the nucleotide sequence shown in SEQ ID NO: 5 (5'-TAGAA-3') are repeated multiple times (5'-(TAGAA) n -3', n is an integer of 2 or more), a base sequence having the base sequence shown in SEQ ID NO: 6 repeated multiple times (5'-(TAAAATAGAA) n (b5) a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 14 (5'-TGGGAATAGAATGGGAA(TAGAA)2(TGGAA)3TAGAATGGAA-3'), the nucleotide sequence shown in SEQ ID NO: 5 (5'-TAGAA-3') are linked in order from the 5' end (5'-(TAGAA) n -3', n is an integer of 2 or more), and a base sequence having the base sequence shown in SEQ ID NO: 6 repeated multiple times (5'-(TAAAATAGAA) n-3', n is an integer of 2 or greater) linked in order from the 5' end. Here, "n" varies depending on the case and is not limited to the following ranges, but examples include integers of 2 to 500, 100 to 400, or 200 to 400.

[0062] The combination of the 5' and 3' regions in the abnormal repeat region of the mutant BEAN1 gene is not particularly limited and may be any combination. The sequences of the 5' and 3' regions are shown in Figure 22.

[0063] In one embodiment, the target binding region contained in the nucleic acid molecule of the present invention may be designed to include the boundary position of two adjacent regions in the non-mutated region adjacent to the 5' side of the 5' region, the 5' region, the core repeat region, the 3' region, and the non-mutated region adjacent to the 3' side of the 3' region. That is, the target binding region contained in the nucleic acid molecule of the present invention may include each of the bases complementary to the terminal bases of the two adjacent regions in the non-mutated region adjacent to the 5' side of the 5' region, the 5' region, the core repeat region, the 3' region, and the non-mutated region adjacent to the 3' side of the 3' region.

[0064] As used herein, a "non-mutated region" refers to a region adjacent to the abnormal repeat region in the locus of a mutant gene containing the abnormal repeat region, which region can also be found in a normal gene. The non-mutated region may be any gene region that can be found in healthy individuals (e.g., individuals not affected by SCA31 or SCA10), and genetic polymorphisms (e.g., single nucleotide polymorphisms found in healthy individuals and polymorphisms related to the number of repeats of a repeat sequence) are permitted. The non-mutated region is either a region adjacent to the 5' side of the 5' region of the abnormal repeat region or a region adjacent to the 3' side of the 3' region of the abnormal repeat region.

[0065] In another embodiment, the entire length of the target binding region contained in the nucleic acid molecule of the present invention is contained in a base sequence complementary to any of the 5' region, the core repeat region, or the 3' region.

[0066] In one embodiment, the nucleic acid molecule of this aspect consists of any one of the base sequences selected from the group consisting of SEQ ID NOs: 156 to 189 and 207 to 209 (wherein u may be t and t may be u).

[0067] In one embodiment, the mutant gene encoding the transcript targeted by the nucleic acid molecule of the present invention is a mutant TK2 gene.

[0068] In the abnormal repeat region that may be contained in the mutant TK2 gene, the core repeat region is a nucleotide sequence (5'-(TGGAA) n -3', n is an integer of 2 or more), i.e., a base sequence in which the base sequence shown in SEQ ID NO: 8 (5'-TTCCA-3') is repeated multiple times (5'-(TTCCA) n −3′; n is an integer of 2 or more). Here, “n” varies depending on the case and is not limited to the following range, but examples include integers of 2 to 500, 100 to 400, or 200 to 400.

[0069] The nucleotide sequences of the 5' and 3' regions in the abnormal repeat region of the mutant TK2 gene are not particularly limited, as long as they are sequences that can be found in patients with spinocerebellar ataxia SCA31.

[0070] The 5' region of the abnormal repeat region of the mutant TK2 gene contains, for example, a sequence in which the nucleotide sequence shown in SEQ ID NO: 16 is repeated multiple times (5'-(TTCTATTTTA) n -3'; n is an integer of 2 or more), and a sequence having multiple repeats of the nucleotide sequence shown in SEQ ID NO: 17 (5'-TTCTA-3') (5'-(TTCTA) n Specific examples of such a base sequence include the following (c1) to (c5): (c1) a base sequence in which the base sequence shown in SEQ ID NO: 16 is repeated multiple times (5'-(TTCTATTTTA) n -3', n is an integer of 2 or more), a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 17 (5'-TTCTA-3') repeated multiple times (5'-(TTCTA) n -3', n is an integer of 2 or more), and a nucleotide sequence shown in SEQ ID NO: 19 (5'-TTCCA(TTCTA)2(TTCCA)2(TTCTA)2TTCCCATTCCATTCCCA-3') linked in order from the 5' end, (c2) a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 16 is repeated multiple times (5'-(TTCTATTTTA) n-3', n is an integer of 2 or more), a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 17 (5'-TTCTA-3') repeated multiple times (5'-(TTCTA) n -3', n is an integer of 2 or more), and a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 21 (5'-TTCCATTCTA(TTCCA)3(TTCTA)2TTCCCATTCTATTCCCA-3') is linked in order from the 5' end, (c3) a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 16 is repeated multiple times (5'-(TTCTATTTTA) n -3', n is an integer of 2 or more), a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 17 (5'-TTCTA-3') repeated multiple times (5'-(TTCTA) n (c3) a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 22 (5'-TTCCATTCTATTCCA(TTCTA)6(TTCCATTCTA)4(TTCCA)3TTCCCATTCTATTCCCA-3') is linked in order from the 5' end, (c4) a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO: 23 (5'-TTCTA(TTTTA)3-3'), the nucleotide sequence shown in SEQ ID NO: 16 is repeated multiple times (5'-(TTCTATTTTA) n -3', n is an integer of 2 or more), a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 17 (5'-TTCTA-3') repeated multiple times (5'-(TTCTA) n -3', n is an integer of 2 or more), and a base sequence in which the base sequence shown in SEQ ID NO: 24 (5'-TTCCA(TTCTA)2TTCCA(TTCTA)2-3') is linked in order from the 5' end, and (c5) a base sequence in which the base sequence shown in SEQ ID NO: 16 is repeated multiple times (5'-(TTCTATTTTA) n -3', n is an integer of 2 or more), a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 17 (5'-TTCTA-3') repeated multiple times (5'-(TTCTA) n and the nucleotide sequence represented by SEQ ID NO: 29 (5'-TTCCATTCTA(TTCCA)3(TTCTA)2TTCCCATTCTATTCCCA-3') linked in order from the 5' end. Here, "n" varies depending on the case and is not limited to the following ranges, but examples include integers of 2 to 500, 100 to 400, or 200 to 400.

[0071] The 3' region in the abnormal repeat region of the mutant TK2 gene includes, for example, a region containing the nucleotide sequence (5'-GTGA-3') shown in SEQ ID NO: 26. Specific nucleotide sequences include the following (d1) to (d4): (d1) the nucleotide sequence (5'-(TTCTA)2TTTTAGTGA-3') shown in SEQ ID NO: 27, (d2) the nucleotide sequence (5'-(TTCTA)4TTTTAGTGA-3') shown in SEQ ID NO: 28, (d3) the nucleotide sequence (5'-GTGA-3') shown in SEQ ID NO: 26, and (d4) a nucleotide sequence (5'-(TTCTA) n and the nucleotide sequence shown in SEQ ID NO: 26 (5'-GTGA-3'; n is an integer of 2 or more) linked in order from the 5' end. Here, "n" varies depending on the case and is not limited to the following ranges, but examples include integers of 2 to 500, 100 to 400, or 200 to 400.

[0072] The combination of the 5' and 3' regions in the abnormal repeat region of the mutant TK2 gene is not particularly limited and may be any combination. The sequences of the 5' and 3' regions are shown in Figure 5.

[0073] In one embodiment, the target binding region contained in the nucleic acid molecule of the present invention may be designed to include the boundary position of two adjacent regions in the non-mutated region adjacent to the 5' side of the 5' region, the 5' region, the core repeat region, the 3' region, and the non-mutated region adjacent to the 3' side of the 3' region. That is, the target binding region contained in the nucleic acid molecule of the present invention may include each of the bases complementary to the terminal bases of the two adjacent regions in the non-mutated region adjacent to the 5' side of the 5' region, the 5' region, the core repeat region, the 3' region, and the non-mutated region adjacent to the 3' side of the 3' region.

[0074] In another embodiment, the entire length of the target binding region contained in the nucleic acid molecule of the present invention is contained in a base sequence complementary to any of the 5' region, the core repeat region, or the 3' region.

[0075] In one embodiment, the nucleic acid molecule of this aspect consists of any one of the base sequences selected from the group consisting of SEQ ID NOs: 33 to 53, 58 to 139, 141 to 150, 152 to 155, and 204 to 206 (however, u in the sequence may be t, and t may be u).

[0076] In one embodiment, the mutant gene encoding the transcript targeted by the nucleic acid molecule of the present invention is a mutant ATXN10 gene.

[0077] In the abnormal repeat region that may be contained in the mutant ATXN10 gene, the core repeat region is a nucleotide sequence (5'-(TGGAA) n -3', n is an integer of 2 or more), i.e., a base sequence in which the base sequence shown in SEQ ID NO: 8 (5'-TTCCA-3') is repeated multiple times (5'-(TTCCA) n −3′; n is an integer of 2 or more). Here, “n” varies depending on the case and is not limited to the following ranges, but examples include integers of 2 to 600, 100 to 500, or 200 to 400.

[0078] The nucleotide sequences of the 5' and 3' regions in the abnormal repeat region of the mutant ATXN10 gene are not particularly limited, as long as they are sequences that can be found in SCA10 spinocerebellar ataxia patients. Specific examples of the 5' region in the abnormal repeat region of the mutant ATXN10 gene include the nucleotide sequence shown in SEQ ID NO: 17 (5'-TTCTA-3') or a nucleotide sequence in which the same is repeated multiple times (5'-(TTCTA) n Specific examples of the 3' region in the abnormal repeat region of a mutant ATXN10 gene include the nucleotide sequence shown in SEQ ID NO: 17 (5'-TTCTA-3'), or a nucleotide sequence in which the nucleotide sequence is repeated multiple times (5'-(TTCTA) n-3', n is an integer of 2 or greater (n is an integer of 2 or greater, and varies depending on the case, and is not limited to the following range, but is, for example, an integer of 100 or greater, 200 or greater, or 400 or greater).

[0079] In one embodiment, the target binding region contained in the nucleic acid molecule of the present invention may be designed to include the boundary position of two adjacent regions in the non-mutated region adjacent to the 5' side of the 5' region, the 5' region, the core repeat region, the 3' region, and the non-mutated region adjacent to the 3' side of the 3' region. That is, the target binding region contained in the nucleic acid molecule of the present invention may include each of the bases complementary to the terminal bases of the two adjacent regions in the non-mutated region adjacent to the 5' side of the 5' region, the 5' region, the core repeat region, the 3' region, and the non-mutated region adjacent to the 3' side of the 3' region.

[0080] In another embodiment, the entire length of the target binding region contained in the nucleic acid molecule of the present invention is contained in a base sequence complementary to any of the 5' region, the core repeat region, or the 3' region.

[0081] In one embodiment, the nucleic acid molecule of this aspect consists of any one of the base sequences selected from the group consisting of SEQ ID NOs: 33 to 37, 58 to 61, 64 to 67, 70 to 139, 141 to 150, 152 to 155, and 204 to 206 (however, u in the sequence may be t, and t may be u).

[0082] The base length of the nucleic acid molecules of the present invention is not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases. The base length of the nucleic acid molecules may be 40 bases or less, 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less. The base length of the nucleic acid molecules may be, for example, 10 to 40 bases, 12 to 30 bases, or 15 to 25 bases. The length can be determined by balancing the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost and synthesis yield. When a nucleic acid such as an aptamer is bound to the nucleic acid molecule, the base length of the nucleic acid molecule as a whole may be the above-mentioned base length plus the base length of the bound nucleic acid.

[0083] The nucleosides contained in the nucleic acid molecules of the present invention may be natural nucleosides (deoxyribonucleosides, ribonucleosides, or both) and / or non-natural nucleosides.

[0084] The nucleic acid molecule of the present invention may be a mixmer. As used herein, the term "mixmer" refers to a nucleic acid chain that contains alternating natural and unnatural nucleosides of periodic or random segment lengths, but does not contain four or more consecutive deoxyribonucleosides and ribonucleosides. A mixmer in which the unnatural nucleoside is a bridged nucleoside and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "BNA / DNA mixmer." A mixmer in which the unnatural nucleoside is a peptide nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "peptide nucleic acid / DNA mixmer." A mixmer in which the unnatural nucleoside is a morpholino nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "morpholino nucleic acid / DNA mixmer." A mixmer is not limited to containing only two types of nucleosides. A mixmer can contain any number of types of nucleosides, whether natural or modified nucleosides or nucleoside mimetics. For example, a mixmer may have one or two consecutive deoxyribonucleosides separated by a bridged nucleoside (e.g., an LNA nucleoside). The bridged nucleoside may further contain a modified nucleobase (e.g., 5-methylcytosine).

[0085] The nucleic acid molecule of the present invention may be a gapmer. As used herein, the term "gapmer" generally refers to a single-stranded nucleic acid consisting of a "central region" (DNA gap region) and wing regions (referred to as the "5' wing region" and "3' wing region," respectively) located directly at the 5' and 3' ends of the central region. In a gapmer, the central region contains at least two (e.g., at least three or at least four) consecutive deoxyribonucleosides (which may contain modified nucleobases recognized by RNase H, such as 5-methylcytosine), and the wing regions contain at least one unnatural nucleoside. Although not limited thereto, unnatural nucleosides contained in the wing regions typically have stronger RNA-binding affinity and higher resistance to nucleases (such as nucleases) than natural nucleosides. The unnatural nucleosides comprising the 5' and 3' wing regions may be, for example, bridged nucleosides and / or 2'-modified nucleosides. When the unnatural nucleosides comprising the wing regions comprise or consist of bridged nucleosides, the gapmer is specifically referred to as a "BNA / DNA gapmer." The number of bridged nucleosides in the 5' and 3' wing regions is at least one, and may be, for example, two or three. The bridged nucleosides in the 5' and 3' wing regions may be contiguous or non-contiguous within the 5' and 3' wing regions. The bridged nucleoside may further comprise a modified nucleobase (e.g., 5-methylcytosine). The bridged nucleoside may be an LNA nucleoside or an ENA nucleoside. When the bridged nucleoside is an LNA nucleoside, the gapmer is specifically referred to as an "LNA / DNA gapmer." When the bridged nucleoside is an ENA nucleoside, the gapmer is referred to as an "ENA / DNA gapmer." When the non-natural nucleosides comprising the 5' and 3' wing regions comprise or consist of peptide nucleic acids, the gapmer is specifically referred to as a "peptide nucleic acid gapmer."When the non-natural nucleosides comprising the 5' wing region and the 3' wing region comprise or consist of morpholino nucleic acids, the gapmer is specifically referred to as a "morpholino nucleic acid gapmer." Furthermore, when the non-natural nucleosides comprising the 5' wing region and the 3' wing region comprise or consist of 2'-modified nucleosides, the 2'-modified group of the 2'-modified nucleoside may be a 2'-O-methyl group or a 2'-O-methoxyethyl group. The number of 2'-modified nucleosides contained in the 5' wing region and the 3' wing region is at least one, and may be, for example, two or three. The 2'-modified nucleosides contained in the 5' wing region and the 3' wing region may be contiguous or non-contiguous within the 5' wing region and the 3' wing region. The 2'-modified nucleoside may further comprise a modified nucleobase (e.g., 5-methylcytosine). When the unnatural nucleosides constituting the 5'-wing region and the 3'-wing region comprise or consist of a bridged nucleoside or a 2'-modified nucleoside, they may be composed of a combination of two or more types of bridged nucleosides and / or 2'-modified nucleosides. For example, combinations of two types may be an LNA nucleoside and an ENA nucleoside; an LNA nucleoside and a 2'-O-methyl nucleoside; an LNA nucleoside and a 2'-O-methoxyethyl nucleoside; an ENA nucleoside and a 2'-O-methyl nucleoside; an ENA nucleoside and a 2'-O-methoxyethyl nucleoside; or a 2'-O-methyl nucleoside and a 2'-O-methoxyethyl nucleoside. For example, a combination of three types may be an LNA nucleoside, an ENA nucleoside, and a 2'-O-methyl nucleoside; an LNA nucleoside, an ENA nucleoside, and a 2'-O-methoxyethyl nucleoside; or an ENA nucleoside, a 2'-O-methyl nucleoside, and a 2'-O-methoxyethyl nucleoside. For example, a combination of four types may be an LNA nucleoside, an ENA nucleoside, a 2'-O-methyl nucleoside, and a 2'-O-methoxyethyl nucleoside.

[0086] When the nucleic acid molecule of the present invention is a gapmer, the DNA gap region may be, for example, 4 to 10 bases long, 5 to 8 bases long, 5 to 8 bases long, 6 to 8 bases long, 7 bases long, or 8 bases long. The DNA gap region is composed of natural nucleosides made of DNA.

[0087] When the nucleic acid molecule of the present invention is a gapmer, the base lengths of the 5' wing region and the 3' wing region of the gapmer may each independently be at least 2 bases long, for example, 2 to 10 bases long, 2 to 7 bases long, 3 to 5 bases long, 3 to 4 bases long, or 3 bases long. The nucleic acid molecule of the present invention may contain at least one LNA or ENA in the 5' wing region and the 3' wing region. For example, the 5' wing region may contain at least one LNA or ENA, for example, 1 to 4, 2 to 4, 2 to 3, or for example, 2. The 3' wing region may contain at least one LNA or ENA, for example, 1 to 4, 2 to 4, 2 to 3, or for example, 2. The type, number, and position of modifications in the 5' wing region and the 3' wing region may be the same or different. The 5' and 3' wing regions may contain a combination of 2'-O-methyl nucleosides, 2'-O-methoxyethyl nucleosides, 2'-LNA, or ENA, and the types of modifications may include one to four, two to three, or, for example, two types, and the types may be the same or different in the 5' and 3' wing regions.

[0088] When the nucleic acid molecule of the present invention is a gapmer, examples of the base lengths of the 5' wing region, DNA gap region, and 3' wing region include 2-8-3, 3-8-2, 3-7-3, 4-6-3, 3-6-4, 4-5-4, 4-7-3, 3-7-4, 4-6-4, 5-6-3, 3-6-5, 3-7-5, 5-7-3, 4-7-4, 4-6-5, 5-6-4, 5-5-5, 5-6-5, etc. Here, in the notation "ABC," "A" indicates the base length of the 5' wing region, "B" indicates the base length of the DNA gap region, and "C" indicates the base length of the 3' wing region.

[0089] In addition, a nucleic acid strand having a wing region only on either the 5'-end or the 3'-end is called a "hemigapmer" in the art, and in this specification, hemigapmers are also included in the term "gapmer."

[0090] The internucleoside linkages in the nucleic acid molecules of the present invention may be naturally occurring internucleoside linkages and / or modified internucleoside linkages. Preferably, at least one, at least two, or at least three internucleoside linkages from the ends (5'-end, 3'-end, or both ends) of the nucleic acid molecules of the present invention are modified internucleoside linkages. Here, for example, the two internucleoside linkages from the end of the nucleic acid chain refer to the internucleoside linkage closest to the end of the nucleic acid chain and the adjacent internucleoside linkage located opposite the end. Modified internucleoside linkages in the terminal region of the nucleic acid chain are preferred because they can suppress or inhibit undesired degradation of the nucleic acid chain. In one embodiment, all or some of the internucleoside linkages in the nucleic acid molecule may be modified internucleoside linkages. The modified internucleoside linkages may be phosphorothioate linkages.

[0091] Nucleic acid molecules of the present invention may comprise, in whole or in part, nucleoside or nucleotide mimetics. Nucleotide mimetics may be peptide nucleic acids and / or morpholino nucleic acids. In one embodiment, nucleic acid molecules of the present invention may comprise or consist of morpholino nucleic acids.

[0092] In one embodiment, less than half or no natural ribonucleosides are present along the entire length of a nucleic acid molecule of the present invention.

[0093] In one embodiment, the nucleic acid molecule of the present invention may comprise modified nucleobases. The number of modified nucleobases is not limited, and may be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6.

[0094] The antisense effect of the nucleic acid molecules of the present invention on target transcripts can be measured by methods known in the art. For example, after introducing the nucleic acid molecules into cells, measurement can be performed using known techniques such as Northern blotting, quantitative PCR, or Western blotting. By measuring the expression level of the target gene or the level of the target transcript (e.g., RNA amount such as mRNA amount, cDNA amount, etc.) in a specific tissue (e.g., the brain), it can be determined whether the nucleic acid molecule suppresses target gene expression in those tissues. A decrease in the measured expression level of the target gene or the level of the target transcript by at least 20%, at least 25%, at least 30%, at least 40%, or at least 50% compared to a negative control (e.g., vehicle administration or no treatment) indicates that the test nucleic acid compound can produce an antisense effect.

[0095] <Double-stranded nucleic acid complex> In one aspect, the present invention relates to a double-stranded nucleic acid complex. The double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand and a second nucleic acid strand. In the double-stranded nucleic acid complex of the present invention, the first nucleic acid strand can function as an ASO. The specific structure of each nucleic acid strand is shown below.

[0096] The first nucleic acid strand can be selected from the embodiments of the nucleic acid molecule described above. For example, the first nucleic acid strand can be a gapmer nucleic acid molecule described above; or the first nucleic acid strand can be a nucleic acid molecule described above that includes: (1) a central region containing at least two (e.g., at least three or at least four) consecutive deoxyribonucleosides; (2) a 5'-wing region located on the 5'-end of the central region and containing an unnatural nucleoside; and (3) a 3'-wing region located on the 3'-end of the central region and containing an unnatural nucleoside. The configurations of the gapmer and the above (1) to (3) are as described above for the nucleic acid molecule.

[0097] The second nucleic acid strand is a nucleic acid molecule containing a base sequence complementary to that of the first nucleic acid strand. In the double-stranded nucleic acid complex of the present invention, the second nucleic acid strand anneals to the first nucleic acid strand through hydrogen bonds of complementary base pairs. Examples of this embodiment include heteroduplex oligonucleotides (HDOs) disclosed in International Publication No. 2013 / 089283, Nishina K, et al., Nature Communication, 2015, 6:7969, and Asami Y, et al., Drug Discoveries & Therapeutics. 2016; 10(5):256-262.

[0098] In one embodiment, the double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand selected from any of the above-described nucleic acid molecule embodiments and a second nucleic acid strand comprising a base sequence complementary to the first nucleic acid strand. In a further embodiment, the second nucleic acid strand may comprise ribonucleosides, deoxyribonucleosides, and / or modified nucleosides.

[0099] In one embodiment, all nucleosides in the region of the second nucleic acid strand consisting of a base sequence complementary to the central region of the first nucleic acid strand may be (a) deoxyribonucleosides; (b) deoxyribonucleosides and ribonucleosides; (c) deoxyribonucleosides and 2'-modified nucleosides; (d) ribonucleosides and 2'-modified nucleosides; or (e) deoxyribonucleosides, ribonucleosides, and 2'-modified nucleosides.

[0100] In one embodiment, the second nucleic acid strand comprises a region comprising at least two consecutive ribonucleosides and / or deoxyribonucleosides complementary to at least two consecutive deoxyribonucleosides in the central region of the first nucleic acid strand. For example, the second nucleic acid strand may comprise a region comprising at least three or at least four consecutive ribonucleosides and / or deoxyribonucleosides complementary to at least three or at least four consecutive deoxyribonucleosides in the central region of the first nucleic acid strand. Here, the number of consecutive deoxyribonucleosides in the central region of the first nucleic acid strand and the number of consecutive ribonucleosides and / or deoxyribonucleosides complementary to the consecutive deoxyribonucleosides in the second nucleic acid strand may be the same or different. For example, the number of consecutive deoxyribonucleosides may be at least four, and the number of consecutive ribonucleosides and / or deoxyribonucleosides may be at least three.

[0101] In a further embodiment, the second nucleic acid strand may include a region consisting of a base sequence complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand. In the second nucleic acid strand, the region consisting of a base sequence complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand may include at least one unnatural nucleoside, which may be, for example, a bridged nucleoside and / or a 2'-modified nucleoside. In a further embodiment, the bridged nucleoside in the second nucleic acid strand is an LNA nucleoside, an ENA nucleoside, or a BNA nucleoside. NC The 2'-modified group of the 2'-modified nucleoside in the second nucleic acid strand may be a 2'-O-methyl group or a 2'-O-methoxyethyl group. When both the first nucleic acid strand and the second nucleic acid strand contain a bridged nucleoside and / or a 2'-modified nucleoside, the bridged nucleosides and / or 2'-modified nucleosides in the first nucleic acid strand and the second nucleic acid strand may be the same or different.

[0102] The internucleoside linkages in the second nucleic acid strand may be naturally occurring internucleoside linkages and / or modified internucleoside linkages. Preferably, at least one, at least two, or at least three internucleoside linkages from the termini (5'-end, 3'-end, or both) of the second nucleic acid strand are modified internucleoside linkages. In one embodiment, all or part of the internucleoside linkages in the second nucleic acid strand may be modified internucleoside linkages. In one embodiment, the second nucleic acid strand may contain modified internucleoside linkages in a region comprising a base sequence complementary to the 5'-wing region and / or the 3'-wing region of the first nucleic acid strand. The modified internucleoside linkages may be phosphorothioate linkages.

[0103] In a further embodiment, the second nucleic acid strand can contain 2'-O-methoxyethyl-modified nucleosides. The number of 2'-O-methoxyethyl-modified nucleosides in the second nucleic acid strand is not limited. For example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or even 100% of the total number of nucleosides in the second nucleic acid strand can be 2'-O-methoxyethyl-modified nucleosides. In one embodiment, all of the nucleosides in the second nucleic acid strand are 2'-O-methoxyethyl-modified nucleosides.

[0104] In further embodiments, the second nucleic acid strand can include one or two or more contiguous 2'-O-methoxyethyl-modified nucleosides at the 5'-terminus and / or one or two or more contiguous 2'-O-methoxyethyl-modified nucleosides at the 3'-terminus. The number of 2'-O-methoxyethyl-modified nucleosides at the 5'-terminus and / or 3'-terminus is not limited. For example, the second nucleic acid strand can include one or two, three, four, five, six, or seven 2'-O-methoxyethyl-modified nucleosides at the 5'-terminus and / or one or two, three, four, five, six, or seven 2'-O-methoxyethyl-modified nucleosides at the 3'-terminus.

[0105] The second nucleic acid strand may also contain 1 to 7 2'-O-methoxyethyl-modified nucleosides at positions other than the one or two or more consecutive 2'-O-methoxyethyl-modified nucleosides located at the 5' end and / or 3' end described above.

[0106] In any of the above embodiments, the types of nucleosides other than 2'-O-methoxyethyl-modified nucleosides in the second nucleic acid strand are not particularly limited. For example, in the second nucleic acid strand, all nucleosides other than 2'-O-methoxyethyl-modified nucleosides may be deoxyribonucleosides.

[0107] In one embodiment, the second nucleic acid strand may comprise modified nucleobases, the number of which is not limited, and may be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6.

[0108] In one embodiment, the second nucleic acid strand may further include at least one overhang region located at one or both of the 5' and 3' ends of the complementary region. An example of this embodiment is described in International Publication No. 2018 / 062510. An "overhang region" refers to a region adjacent to a complementary region, where, when the first and second nucleic acid strands anneal to form a double-stranded structure, the 5' end of the second nucleic acid strand extends beyond the 3' end of the first nucleic acid strand and / or the 3' end of the second nucleic acid strand extends beyond the 5' end of the first nucleic acid strand, i.e., a nucleotide region in the second nucleic acid strand that protrudes from the double-stranded structure. The overhang region in the second nucleic acid strand may be located at either the 5' end or the 3' end of the complementary region. The overhang region in the second nucleic acid strand may be located at either the 5' end or the 3' end of the complementary region.

[0109] In one embodiment, a functional moiety may be bound to the first nucleic acid strand and / or the second nucleic acid strand, for example, the second nucleic acid strand. The bond between the first nucleic acid strand and / or the second nucleic acid strand and the functional moiety may be a direct bond or an indirect bond via another substance, but in one embodiment, the first nucleic acid strand and / or the second nucleic acid strand and the functional moiety are preferably directly bound to each other via a covalent bond, an ionic bond, a hydrogen bond, or the like, and a covalent bond is more preferred from the viewpoint of obtaining a more stable bond.

[0110] In certain embodiments, the structure of the "functional moiety" is not particularly limited, and it confers a desired function to the double-stranded nucleic acid complex to which it is bound. Desired functions include labeling, purification, and target delivery. Examples of moieties that impart labeling include compounds such as fluorescent proteins and luciferase. Examples of moieties that impart purification include compounds such as biotin, avidin, His-tag peptides, GST-tag peptides, and FLAG-tag peptides. Furthermore, from the viewpoint of highly specific and efficient delivery of the first nucleic acid strand to a target site and highly effective suppression of target gene expression by the nucleic acid, it is preferable that a molecule having the activity of delivering the double-stranded nucleic acid complex to a target site be bound as a functional moiety to the first nucleic acid strand and / or the second nucleic acid strand in certain embodiments. Examples of moieties that impart target delivery function include lipids, antibodies, aptamers, and ligands for specific receptors.

[0111] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand, for example, the second nucleic acid strand, is bound to a lipid. Examples of the lipid include, but are not limited to, tocopherol, cholesterol, fatty acids, phospholipids and their analogs; folic acid, vitamin C, vitamin B1, vitamin B2; estradiol, androstane and their analogs; steroids and their analogs; ligands for LDLR, SRBI, or LRP1 / 2; FK-506, and cyclosporine; and lipids described in PCT / JP2019 / 12077 and PCT / JP2019 / 10392. The lipid may be tocopherol or an analog thereof and / or cholesterol or an analog thereof, substituted or unsubstituted C １～３０an alkyl group of the formula ２～３０ or a substituted or unsubstituted alkenyl group of １～３０ In one embodiment, the second nucleic acid strand may be bound to tocopherol or cholesterol or an analog thereof.

[0112] The functional moiety may be linked to the 5'-end, the 3'-end, or both ends of the first and / or second nucleic acid strand. Alternatively, the functional moiety may be linked to an internal nucleotide of the first and / or second nucleic acid strand. The first and / or second nucleic acid strand may contain two or more functional moieties, such as lipids, which may be linked to multiple positions on the first and / or second nucleic acid strand and / or may be linked as a group to a single position on the first and / or second nucleic acid strand. One functional moiety may be linked to the 5'-end and one to the 3'-end of the first and / or second nucleic acid strand.

[0113] The bond between the first and / or second nucleic acid strand and the functional moiety may be a direct bond or an indirect bond mediated by another substance. However, in certain embodiments, the functional moiety is preferably directly bonded to the first and / or second nucleic acid strand via a covalent bond, ionic bond, hydrogen bond, etc., and a covalent bond is more preferred in view of obtaining a more stable bond.

[0114] The functional moiety may also be linked to the first nucleic acid strand and / or the second nucleic acid strand via a cleavable or noncleavable linker. In this case, the first nucleic acid strand and the second nucleic acid strand may be linked via a linker to form a single strand. However, since the functional region in this case has the same structure as in the double-stranded nucleic acid complex, this specification also encompasses such single-stranded nucleic acids as an embodiment of the double-stranded nucleic acid complex of the present invention. The linker may be any polymer. Examples include polynucleotides, polypeptides, and alkylenes. Specifically, it may be composed of natural nucleotides such as DNA and RNA, or unnatural nucleotides such as peptide nucleic acids and morpholino nucleic acids. When the linker is composed of a nucleic acid, the chain length of the linker may be at least one base, for example, 3 to 10 bases or 4 to 6 bases. A chain length of 4 bases is preferred. In this case, the linker may take the form of a hinge (hairpin loop). The linker can be located on either the 5' or 3' side of the first nucleic acid strand. For example, when the second nucleic acid strand is bound to the 5' side of the first nucleic acid strand, the 5' end of the first nucleic acid strand and the 3' end of the second nucleic acid strand are linked via the linker.

[0115] "Cleavable linker" refers to a linking group that is cleaved under physiological conditions, e.g., within a cell or an animal (e.g., within a human body). In certain embodiments, the cleavable linker is selectively cleaved by an endogenous enzyme, such as a nuclease. Cleavable linkers include amide, ester, phosphodiester or both esters, phosphate ester, carbamate, and disulfide bonds, as well as natural DNA linkers.

[0116] The term "non-cleavable linker" refers to a linker that is not cleaved under physiological conditions, for example, within a cell or an animal body (e.g., within the human body). Examples of non-cleavable linkers include, but are not limited to, linkers consisting of phosphorothioate bonds, and modified or unmodified deoxyribonucleosides or modified or unmodified ribonucleosides linked by phosphorothioate bonds. When the linker is a nucleic acid such as DNA or an oligonucleotide, the chain length is not particularly limited, but may typically be 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases.

[0117] A specific example of the linker is a linker represented by the following formula (I):

[0118] (In the formula, L 2 is a substituted or unsubstituted C1 to C 12 (e.g., propylene, hexylene, dodecylene), a substituted or unsubstituted C3 to C8 cycloalkylene group (e.g., cyclohexylene), —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, or CH(CH2-OH)—CH2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-; L 3 represents -NH- or a bond, and L 4 is a substituted or unsubstituted C1 to C 12 alkylene groups (e.g., ethylene, pentylene, heptylene, undecylene), substituted or unsubstituted C3-C8 cycloalkylene groups (e.g., cyclohexylene), -(CH2)2-[O-(CH2)2] m - or a bond, where m is an integer from 1 to 25; L 5 represents -NH-(C=O)-, -(C=O)-, or a bond (wherein the substitution is preferably made by a halogen atom).

[0119] In one embodiment, the linker of formula (I) is L 2is an unsubstituted C3 to C6 alkylene group (e.g., propylene, hexylene), —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, or —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, and L 3 is -NH-, and L 4 and L 5 is a bond.

[0120] The base lengths of the first and second nucleic acid strands are not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases. The base lengths of the first and second nucleic acid strands may be 40 bases or less, 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less. The first and second nucleic acid strands may be the same length or different lengths (e.g., one of them may be 1 to 3 bases shorter or longer). The double-stranded structure formed by the first and second nucleic acid strands may include a bulge. The length can be selected based on the balance between the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost and synthesis yield. When a nucleic acid such as an aptamer is bound to the first nucleic acid strand and / or the second nucleic acid strand, the total base length of the first nucleic acid strand and the second nucleic acid strand may be the above-mentioned base length plus the base length of the bound nucleic acid. In this case, the base length of the bound nucleic acid is not limited, but may be, for example, at least 10 bases, at least 15 bases, or at least 20 bases, or may be 100 bases or less, 80 bases or less, 60 bases or less, 40 bases or less, or 30 bases or less.

[0121] In one embodiment, the mutant gene encoding the transcript targeted by the double-stranded nucleic acid complex of the present invention is a mutant TK2 gene, the base sequence of the first nucleic acid strand consists of the base sequence set forth in SEQ ID NO: 65 (however, t in the sequence may also be u), and the base sequence of the second nucleic acid strand consists of the base sequence set forth in SEQ ID NO: 140, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, or SEQ ID NO: 203 (however, t in the sequence may also be u). In this embodiment, the first nucleic acid strand is not particularly limited in terms of sugar modifications or internucleoside modifications, so long as its base sequence consists of the base sequence set forth in SEQ ID NO: 65 (however, t in the sequence may also be u). For example, all or part of the internucleoside linkages may be modified internucleoside linkages, and the modified internucleoside linkages may be phosphorothioate linkages. The same applies to the second nucleic acid strand.

[0122] In a further embodiment, the mutant gene encoding the transcript targeted by the double-stranded nucleic acid complex of the present invention is a mutant TK2 gene, the first nucleic acid strand consists of the nucleic acid strand set forth in SEQ ID NO: 65, and the second nucleic acid strand consists of the nucleic acid strand set forth in SEQ ID NO: 140, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, or SEQ ID NO: 203. The base sequence, sugar modifications, and internucleoside linkages of the first nucleic acid strand in this embodiment are all specified as the structure set forth in SEQ ID NO: 65. The same applies to the second nucleic acid strand.

[0123] <Pharmaceutical Composition> In one aspect, the present invention relates to a pharmaceutical composition, which contains the nucleic acid molecule or double-stranded nucleic acid complex described herein as an active ingredient and can be used to treat spinocerebellar ataxia type 31 (SCA31) or spinocerebellar ataxia type 10 (SCA10).

[0124] (Active Ingredient) The pharmaceutical composition of the present invention may contain two or more types of nucleic acid molecules and / or double-stranded nucleic acid complexes described herein.

[0125] In one embodiment, the present invention provides a pharmaceutical composition for treating spinocerebellar ataxia type 31 (SCA31), comprising a nucleic acid molecule and / or a double-stranded nucleic acid complex targeting a transcription product of a mutant BEAN1 gene.

[0126] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating spinocerebellar ataxia type 31 (SCA31), comprising a nucleic acid molecule and / or a double-stranded nucleic acid complex targeting a transcription product of a mutant TK2 gene.

[0127] In one embodiment, the present invention provides a pharmaceutical composition for treating spinocerebellar ataxia type 31 (SCA31), comprising a nucleic acid molecule and / or double-stranded nucleic acid complex targeting a transcription product of a mutant BEAN1 gene, and a nucleic acid molecule and / or double-stranded nucleic acid complex targeting a transcription product of a mutant TK2 gene.

[0128] In one embodiment, the present invention provides a pharmaceutical composition for treating spinocerebellar ataxia type 10 (SCA10), comprising a nucleic acid molecule and / or a double-stranded nucleic acid complex targeting a transcription product of a mutant ATXN10 gene.

[0129] The amount (content) of nucleic acid molecules or double-stranded nucleic acid complexes contained in a pharmaceutical composition varies depending on the type of nucleic acid molecule or double-stranded nucleic acid complex, the site of delivery (e.g., the brain), the dosage form of the pharmaceutical composition, the dosage of the pharmaceutical composition, and the type of carrier (described below). Therefore, it can be determined appropriately taking into account each condition. Typically, a single dose of pharmaceutical composition is adjusted to contain an effective amount of nucleic acid molecules or double-stranded nucleic acid complexes. The term "effective amount" refers to the amount of nucleic acid molecules or double-stranded nucleic acid complexes necessary to function as active ingredients. An "effective amount" may be an amount that causes little or no harmful side effects to the organism to which it is applied. This effective amount may vary depending on various conditions, such as subject information, administration route, and administration frequency. Ultimately, it is determined by the judgment of a physician, veterinarian, or pharmacist. "Subject information" refers to various individual information about the organism to which the pharmaceutical composition is applied. For example, if the subject is a human, this information includes age, weight, sex, diet, health condition, disease progression and severity, drug sensitivity, and the presence or absence of concomitant medications.

[0130] The pharmaceutical composition of the present invention may consist solely of the nucleic acid molecule or double-stranded nucleic acid complex described herein, or may further contain auxiliary components such as a carrier in addition to the nucleic acid molecule or double-stranded nucleic acid complex described herein.

[0131] (Carrier) The pharmaceutical composition of the present invention can contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to an additive commonly used in the pharmaceutical technology field. Examples include solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavors, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonicity adjusters, sedatives, bulking agents, disintegrants, buffers, coating agents, lubricants, colorants, sweeteners, thickeners, flavoring agents, solubilizers, and other additives.

[0132] The solvent may be, for example, water or any other pharmaceutically acceptable aqueous solution, or a pharmaceutically acceptable organic solvent. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.

[0133] The above-mentioned carriers are used to avoid or suppress the degradation of the active ingredient, the nucleic acid molecule or double-stranded nucleic acid complex, by enzymes and the like in the body, as well as to facilitate formulation and administration methods and maintain the dosage form and medicinal efficacy, and may be used appropriately as needed.

[0134] (Dosage Form) The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a form that can exert the pharmacological effect of the active ingredient in vivo without inactivating the active ingredient, the nucleic acid molecule or double-stranded nucleic acid complex described in this specification, by degradation or the like.

[0135] The specific dosage form varies depending on the administration method and / or formulation conditions. The administration method can be roughly divided into parenteral administration and oral administration, and therefore, a dosage form suitable for each administration method may be used.

[0136] If the administration method is parenteral administration, the preferred dosage form is a liquid that can be administered directly to the target site or systemically via the circulatory system. Examples of liquids include injections. Injections can be formulated by appropriately combining the above-mentioned excipients, elixirs, emulsifiers, suspending agents, surfactants, stabilizers, pH adjusters, etc., and mixing them in a unit dosage form required for generally accepted pharmaceutical practice. Other forms include ointments, plasters, cataplasms, transdermal agents, lotions, inhalants, aerosols, eye drops, and suppositories.

[0137] When the administration method is oral administration, preferred dosage forms include solids (including tablets, capsules, drops, and lozenges), granules, powders, powders, and liquids (including oral solutions, emulsions, and syrups). If a solid is used, it can be made into a dosage form coated with a coating known in the art, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multi-layer tablets, as needed.

[0138] The specific shape and size of each of the above dosage forms are not particularly limited as long as they are within the range of dosage forms known in the art. The pharmaceutical composition of the present invention may be formulated according to a conventional method in the art.

[0139] (Dosage form and dosage) In the present specification, there is no particular limitation on the preferred administration form of the pharmaceutical composition. Administration may be systemic administration or local administration. The administration route may be oral administration or parenteral administration. Specific examples of parenteral administration include intravenous administration, intraarterial administration, administration by blood transfusion, intraperitoneal administration, intraventricular administration, intrathecal administration, intraocular administration, intramuscular administration, subcutaneous administration (including implantable continuous subcutaneous administration), intradermal administration, intravesical administration, intravaginal administration, rectal administration, inhalation or nasal instillation administration, and tracheal / bronchial administration. When the target site of application of the present invention is the brain, intraventricular administration or intrathecal administration, which are the target sites, is preferred.

[0140] When the pharmaceutical composition is administered or ingested, the dosage or intake may be, for example, such that the nucleic acid molecule or double-stranded nucleic acid complex contained therein is 0.00001 mg / kg / day to 10,000 mg / kg / day, or 0.001 mg / kg / day to 100 mg / kg / day. The pharmaceutical composition may be administered in a single dose or multiple doses. In the case of multiple doses, the composition may be administered daily or at appropriate intervals (e.g., at intervals of 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month), for example, 2 to 20 times. The single dose of the nucleic acid molecule or double-stranded nucleic acid complex can be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 0.1 mg / kg or more, 0.25 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2.5 mg / kg or more, 0.5 mg / kg or more, 1.0 mg / kg or more, 2.0 mg / kg or more, 3.0 mg / kg or more, 4.0 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more, for example, 0.001 mg / kg to 500 mg / kg. Any amount within the mg / kg range (e.g., 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg) can be appropriately selected.

[0141] The nucleic acid molecule or double-stranded nucleic acid complex of the present invention may be administered twice a week for four doses at a dose of 0.01 to 10 mg / kg (e.g., about 6.25 mg / kg). Alternatively, the nucleic acid molecule or double-stranded nucleic acid complex may be administered once or twice a week for two to four doses at a dose of 0.05 to 30 mg / kg (e.g., about 25 mg / kg), for example, twice a week for two doses. The use of such a dosing regimen (divided administration) can reduce toxicity (e.g., avoid platelet reduction) and alleviate the burden on the subject compared to a single administration of a higher dose.

[0142] The pharmaceutical composition exerts an additive inhibitory effect within cells even when administered repeatedly. Furthermore, when administering repeatedly, the efficacy can be improved by leaving a certain interval between administrations (e.g., half a day or more).

[0143] The present invention also provides nucleic acid molecules and / or double-stranded nucleic acid complexes targeting the transcription product of the mutant BEAN1 gene described herein, and / or nucleic acid molecules and / or double-stranded nucleic acid complexes targeting the transcription product of the mutant TK2 gene, for use in the treatment of spinocerebellar ataxia type 31 (SCA31).

[0144] The present invention also provides nucleic acid molecules and / or double-stranded nucleic acid complexes that target the transcription products of the mutant ATXN10 gene described herein for use in treating spinocerebellar ataxia type 10 (SCA10).

[0145] <Method> In one aspect, the present invention relates to a method for producing an antisense effect on a transcription product, comprising administering to a subject a nucleic acid molecule, a double-stranded nucleic acid complex, or a pharmaceutical composition described herein. The method may be a method for treating or preventing a disease in a subject.

[0146] <Therapeutic Methods> In one aspect, the present invention relates to a therapeutic method comprising administering to a patient an effective amount of a nucleic acid molecule, double-stranded nucleic acid complex, or pharmaceutical composition described herein. The disease targeted by the therapeutic method of this aspect is spinocerebellar ataxia type 31 (SCA31) or spinocerebellar ataxia type 10 (SCA10). The method for treating spinocerebellar ataxia type 31 (SCA31) comprises administering to a patient an effective amount of a nucleic acid molecule and / or double-stranded nucleic acid complex targeting a transcript of a mutant BEAN1 gene described herein, and / or a nucleic acid molecule and / or double-stranded nucleic acid complex targeting a transcript of a mutant TK2 gene described herein. The method for treating spinocerebellar ataxia type 10 (SCA10) comprises administering to a patient an effective amount of a nucleic acid molecule and / or double-stranded nucleic acid complex targeting a transcript of a mutant ATXN10 gene described herein.

[0147] <Production Method> Those skilled in the art can produce the nucleic acid molecule, double-stranded nucleic acid complex, or pharmaceutical composition of the present invention by appropriately selecting a known method. Although not limited thereto, the process typically begins with designing and producing each of the first and second nucleic acid strands that make up the nucleic acid molecule or double-stranded nucleic acid complex. For example, the nucleic acid molecule or first nucleic acid strand is designed based on the base sequence information of the target transcription product (e.g., the base sequence of the target gene), and the second nucleic acid strand is designed as its complementary strand. Next, based on the designed base sequence information, each nucleic acid strand can be synthesized using the phosphoramidite method using a commercially available automated nucleic acid synthesizer, for example, from GE Healthcare, Thermo Fisher Scientific, Beckman Coulter, etc. The resulting oligonucleotides can then be purified using a reverse-phase column, ion exchange column, etc.

[0148] Furthermore, in the case of a double-stranded nucleic acid complex to which a functional moiety is bound, the first nucleic acid strand may be produced according to the above-described method. Meanwhile, the second nucleic acid strand to which a functional moiety is bound can be produced by carrying out the above-described synthesis and purification using a nucleic acid species to which a functional moiety has already been bound. For example, the second nucleic acid strand may be produced by carrying out the above-described synthesis and purification using a nucleic acid species to which a functional moiety has already been bound. Alternatively, a functional moiety may be bound to the second nucleic acid strand produced by carrying out the above-described synthesis and purification using a known method. After producing each nucleic acid strand, the first nucleic acid strand and the second nucleic acid strand are annealed as described below to produce a double-stranded nucleic acid complex to which the desired functional moiety is bound.

[0149] Methods for linking functional moieties to nucleic acids are well known in the art. Nucleic acids prepared by this method can be mixed in an appropriate buffer solution and denatured at about 90°C to 98°C for several minutes (e.g., 5 minutes). The nucleic acids are then annealed at about 30°C to 70°C for about 1 to 8 hours to produce one of the double-stranded nucleic acid complexes of the present invention. Alternatively, nucleic acid strands can be ordered from various manufacturers (e.g., Gene Design, Inc.) by specifying the base sequence and the modification site and type. The annealing step can be carried out by allowing the solution to stand at room temperature (about 10°C to 35°C) for about 5 to 60 minutes. The first and second nucleic acid strands can be dissolved in a buffer solution (e.g., phosphate-buffered saline) or water at about 70°C to 98°C, respectively, and the resulting two solutions can be mixed. The mixture can be maintained at about 70°C to 98°C for several minutes (e.g., 5 minutes), followed by maintaining the mixture at about 30°C to 70°C (or about 30°C to 50°C) for about 1 to 8 hours to prepare the double-stranded nucleic acid complex of some embodiments of the present invention. The first nucleic acid strand and the second nucleic acid strand can each be dissolved in a buffer solution (e.g., phosphate-buffered saline) or water at room temperature (about 10°C to 35°C). The annealing conditions (time and temperature) for preparing the double-stranded nucleic acid complex are not limited to those described above. Conditions suitable for promoting annealing of nucleic acid strands are well known in the art.

[0150] In one embodiment, there is provided use of a nucleic acid molecule and / or double-stranded nucleic acid complex targeting a transcription product of a mutant BEAN1 gene described herein, and / or a nucleic acid molecule and / or double-stranded nucleic acid complex targeting a transcription product of a mutant TK2 gene, for use in the treatment of spinocerebellar ataxia type 31 (SCA31).

[0151] In another embodiment, there is provided the use of a nucleic acid molecule and / or a double-stranded nucleic acid complex targeting a transcription product of a mutant ATXN10 gene described herein for use in treating spinocerebellar ataxia type 10 (SCA10).

[0152] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0153] Example 1: Synthesis of ASO (LNA / DNA gapmer) targeting SCA31 mutant repeat (TK2) Antisense oligonucleotides (ASO) targeting the SCA31 abnormal repeat region in the mutant TK2 gene transcript (hereinafter referred to as "SCA31 mutant repeat (TK2)") were synthesized by Gene Design Inc. (Osaka, Japan).

[0154] The synthesized ASOs are shown in Table 1 and Figure 6. All of the ASOs synthesized in this example were LNA / DNA gapmers.

[0155]

[0156] <Example 2: Verification of the gene expression inhibitory effect of the LNA / DNA gapmer synthesized in Example 1> (Purpose) To verify the gene expression inhibitory effect of the ASO (LNA / DNA gapmer) synthesized in Example 1 on the SCA31 mutant repeat (TK2).

[0157] (Methods and Results) (1) qRT-PCR The SCA31 mutant repeat (TK2) was transiently expressed and the ASO prepared in Example 1 was introduced into HeLa cells to verify the effect of suppressing gene expression of the SCA31 mutant repeat (TK2).

[0158] For transient expression of the SCA31 mutant repeat (TK2), we used the pkSCX-IRES-EGFP vector, which contains the SCA31 mutant repeat (TK2) sequence downstream of the chicken β-actin promoter and an IRES-EGFP sequence immediately following it (Figure 36A). 0.8 μg of the pkSCX-IRES-EGFP vector was diluted in 50 μL of Opti-MEM. A 10 μM ASO stock solution was prepared in PBS.

[0159] In the examples herein, unless otherwise specified, PBS was used as a negative control (shown as "Control" in the figures), and the same amount of PBS as the ASO to be introduced was added during gene introduction.

[0160] In addition, an ASO consisting of a base sequence unrelated to the target sequence was used as an additional control (shown as "Unrelated" in the figure), and one of the sequences shown in Table 2 below was used.

[0161]

[0162] ASO (0.1-2 nM) was transfected onto 1.2 x 10 HeLa cells using 50 μL of Opti-MEM containing 2 μL of Lipofectamin 2000 along with the pkSCX-IRES-EGFP vector containing the SCA31 insert in the TK2 orientation (Figure 36A). 5 HeLa cells were co-transfected with SCA31-0 at a concentration of 1000 / µL. 24 hours after transfection, RNA was recovered from HeLa cells by extraction with ISOGEN and isopropanol precipitation. 250 ng of the recovered RNA was then converted to cDNA using PrimeScript RT Reagent (Takara Bio). Using this stock solution as a template, qRT-PCR was performed using a Roche Light Cycler 480II with a probe primer mixture consisting of SCA31-0_primer FW (SEQ ID NO: 18, 5'-TGGCTGCACATAGCTTTATCTCTT-3') and SCA31-0_primer RV (SEQ ID NO: 20, 5'-AAGCCCAATCTGGAAGCAAA-3'). The position detected by qRT-PCR is shown as "SCA31-0" in Figure 36A. ACTB (Hs01060665-g1, ABI) was used as a reference, and individual results were analyzed using the ΔΔCt method, and significance was analyzed using the t-test.

[0163] To confirm the transduction efficiency, ds-Red was co-transduced at 1 / 40 the amount of the pkScx-IRES-EGFP vector, and it was confirmed that there was no difference in fluorescence intensity 24 hours later.

[0164] The results of qRT-PCR are shown in Figure 7. In particular, T-j1, T-j2, T-r2, T-r1, T-r3, T-r4, T-r5, T-r8, T-r7, T-r12, T-r13, T-r16, and T-r10 exerted a strong inhibitory effect on the SCA31 mutant repeat (TK2). These results suggest that a strong inhibitory effect tends to be observed when the ASO target sequence includes the boundary between the 5' region and the core repeat region, the boundary between the core repeat region and the 3' region, or when the ASO target sequence is located within the core repeat region.

[0165] (2) FISH From the ASOs used in (1) above, T-r1 (SEQ ID NO: 33) was selected and its gene expression suppression effect on the SCA31 mutant repeat (TK2) was verified by FISH. Similar verification was also performed for other sequences.

[0166] HeLa cells were transfected with the ASO (T-r1) along with the pkSCX-IRES-EGFP vector containing the SCA31 insert in the TK2 orientation, as described above (1). 24 hours after transfection, the cells were washed with 1x PBS for 5 minutes and prefixed with 4% PFA for 1 hour. After a 5-minute wash with 1x PBS, the cells were incubated with 0.2N HCl for 20 minutes to disrupt the cell wall, and then permeabilized with 0.1% Triton X-100 for 10 minutes. After a 5-minute wash with 1x PBS and postfixation with 4% PFA for 5 minutes, the cells were acetylated with triethanolamine acetic anhydride for 20 minutes. After two 5-minute washes with 4x SSC and a 30-minute prehybridization, the cells were hybridized with a DIG-labeled LNA-(TGGAA)5 probe at 60°C for 2 hours. The sections were washed at 60°C for 5 minutes in 4x SSC, 3 times for 20 minutes in 2x SSC / Formamide, and 3 times for 40 minutes in 0.1x SSC. After blocking for 30 minutes, they were incubated with a 1 / 2000 dilution anti-DIG-AP antibody at 4°C overnight. After washing four times for 15 minutes in TBS-t, they were incubated for 10 minutes in detection solution (0.1M Tris-HCl, 10mM MgCl2, 0.1M NaCl) and then developed with HNPP / FastRed for 30 minutes. Finally, nuclei were stained with Hoechst for 10 minutes and embedded in VECTASHEILD.

[0167] The results of FISH are shown in Figure 8. In Figure 8, the arrow indicates cells in which expression of the SCA31 mutant repeat (TK2) was detected. It was demonstrated that the expression of the SCA31 mutant repeat (TK2) was suppressed by ASO (T-r1).

[0168] Example 3: Synthesis of ENA / DNA gapmer targeting SCA31 mutant repeat (TK2) In this example, the synthesis method of ASO targeting SCA31 mutant repeat (TK2) was performed using T-re10: HO-T e2s -G e2s -G s -A s -A s -T s -G s -G s -A s -A s -T e2s -G e2s -G 2t -H (SEQ ID NO: 59) (the meanings of the symbols in the formula are as described above) will be used as an example.

[0169] Synthesis was performed using an automated nucleic acid synthesizer (BioAutomation MerMade 192X) using the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984)). The reagents used were activator solution-3 (0.25 mol / L 5-benzylthio-1H-tetrazole in acetonitrile, Wako Pure Chemical Industries, Ltd., product No. 013-20011), CAP A for AKTA (1-methylimidazole in acetonitrile, Sigma-Aldrich, product No. L040050), Cap B1 for AKTA (acetic anhydride in acetonitrile, Sigma-Aldrich, product No. L050050), Cap B2 for AKTA (pyridine in acetonitrile, Sigma-Aldrich, product No. L050150), DCA Deblock (dichloroacetic acid in toluene, Sigma-Aldrich, product No. The thiolation reagent used to form phosphorothioate bonds was phenylacetyl disulfide (CARBOSYNTH, product No. FP07495) dissolved in a 1:1 (v / v) solution of acetonitrile (anhydrous, Kanto Chemical, product No. 01837-05) and pyridine (anhydrous, Kanto Chemical, product No. 11339-05). The amidite reagents used were DNA phosphoramidites (adenosine product No. ANP-5551, guanosine product No. ANP-5553, thymidine product No. ANP-5554) and 2'-OMe nucleoside phosphoramidites (adenosine product No. ANP-5751, cytidine product No. ANP-5752, guanosine product No. ANP-5753, The uridine derivative (product No. ANP-5754) was manufactured by ChemGenes.The 5-methyldeoxycytidine phosphoramidite used was compound 6f from the reference (Organic Process Research & Development, 2000, 4, 175-181). Non-natural phosphoramidites include those described in JP-A 2000-297097, such as Example 14 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-6-N-benzoyladenosine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite), Example 27 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-2-N-isobutyrylguanosine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite), and Example 22 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite). The compounds used were those from Example 1 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite) and Example 2 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite). The solid support used was Glen Unysupport FC 96-well format 0.2 μmol (GlenResearch), and the above T-re10 (SEQ ID NO: 59) was synthesized. The condensation time for the amidite was approximately 9 minutes.

[0170] The protected oligonucleotide analogues containing the target sequence were treated with 600 μL of concentrated aqueous ammonia to cleave the oligomer from the support and remove the cyanoethyl protecting groups on the phosphorus atom and the nucleobases. The oligomer mixture was mixed with 300 μL of Clarity QSP DNA Loading Buffer (Phenomenex) and loaded onto a Clarity SPE 96-well plate (Phenomenex). 1 mL of a 1:1 Clarity QSP DNA Loading Buffer:water solution, 3 mL of water, 3 mL of 3% dichloroacetic acid (DCA) solution, and 6 mL of water were added in that order. The components extracted with a 9:1 20 mM Tris aqueous solution:acetonitrile solution were then collected. The target compound was obtained after evaporation. This compound was analyzed by reverse-phase HPLC (column (Phenomenex, Clarity 2.6 μm Oligo-MS 100A (2.1 × 50 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM triethylamine aqueous solution, solution B: methanol, B%: 10% → 25% (4 min, linear gradient; 60 °C; 0.5 mL / min; 260 nm)), eluting at 2.12 min. The compound was identified by negative ion electrospray ionization mass spectrometry.

[0171] The synthesized ASOs are shown in Table 3. All of the ASOs synthesized in this example were ENA / DNA gapmers.

[0172]

[0173] <Example 4: Verification of the gene expression suppression effect of the ASO (ENA / DNA gapmer) synthesized in Example 3> (Purpose) To verify the gene expression suppression effect of the ASO (ENA / DNA gapmer) synthesized in Example 3 on the SCA31 mutant repeat (TK2).

[0174] (Method and Results) ASOs were transfected into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the TK2 direction, and the expression level of the SCA31 mutant repeat (TK2) was quantified by qRT-PCR using the same method as in Example 2. The LNA / DNA gapmer synthesized in Example 1 and the ENA / DNA gapmer synthesized in Example 3 were used as ASOs at concentrations of 0.1 nM, 0.5 nM, or 2 nM.

[0175] The results of quantifying the expression level of the SCA31 mutant repeat (TK2) are shown in Figure 9. These results demonstrate that the ENA / DNA gapmer has an inhibitory effect on expression equivalent to that of the LNA / DNA gapmer.

[0176] <Example 5: ASO targeting SCA31 mutant repeat (TK2): Gapmer with ENA-2'-OMe RNA-ENA wing region> (Purpose) An ENA / DNA gapmer with an ENA-2'-OMe RNA-ENA structure in the wing region is synthesized to verify its effect of suppressing gene expression of SCA31 mutant repeat (TK2).

[0177] (Method and Results) ASOs were synthesized using the same method as in Example 3. The synthesized ASOs are shown in Table 4. All ASOs synthesized in this example were ENA / DNA gapmers with ENA-2'-OMe RNA-ENA wing regions.

[0178]

[0179] The ASO was transfected into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence oriented in the TK2 direction, and the expression level of the SCA31 mutant repeat (TK2) was quantified by qRT-PCR using the same method as in Example 2. The ASO was used at concentrations of 0.1 nM, 0.5 nM, 1 nM, or 2 nM.

[0180] The results of quantifying the expression level of the SCA31 mutant repeat (TK2) are shown in Figure 10. These results demonstrate that the ENA / DNA gapmer with the ENA-2'-OMe RNA-ENA wing region can suppress expression to a similar extent as the LNA / DNA gapmer.

[0181] <Example 6: ASO targeting SCA31 mutant repeat (TK2): surrounding sequence of T-re10a> (Purpose) An ASO consisting of the surrounding sequence of T-re10a, which showed a strong expression-suppressing effect on the SCA31 mutant repeat (TK2) in Example 5, will be synthesized and its effect will be verified.

[0182] (Method and Results) ASO was synthesized using the same method as in Example 3. The synthesized ASO is shown in Table 5.

[0183]

[0184] Using the same method as in Example 2, ASO (0.1 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the TK2 direction, and the expression level of the SCA31 mutant repeat (TK2) was quantified by qRT-PCR.

[0185] The results of quantifying the expression level of the SCA31 mutant repeat (TK2) are shown in Figure 11. Among the ASOs listed in Table 5, T-re10e exhibited an expression-suppressing effect equivalent to that of T-re10a. T-re10f to T-re10p also exhibited an expression-suppressing effect. Of these compounds, T-re10a and T-re10e were found to have the strongest expression-suppressing effect.

[0186] Example 7: ENA / DNA gapmer targeting the core repeat region of the SCA31 mutant repeat (TK2) Objective: To synthesize an ENA / DNA gapmer targeting the core repeat region of the SCA31 mutant repeat (TK2) and having an ENA-2'-OMe RNA-ENA structure in the wing region, and to verify its effect on suppressing gene expression of the SCA31 mutant repeat (TK2).

[0187] (Method and Results) ASOs were synthesized using the same method as in Example 3. The synthesized ASOs are shown in Table 6. The position of each ASO in the SCA31 mutation repeat (TK2) is also shown in Figure 12.

[0188]

[0189] Using the same method as in Example 2, ASO (0.1 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the TK2 direction, and the expression level of the SCA31 mutant repeat (TK2) was quantified by qRT-PCR.

[0190] The results of quantifying the expression level of the SCA31 mutant repeat (TK2) are shown in Figure 13. T-r1a, T-r2a, T-r2b, T-r2c, and T-r2d all showed strong gene expression suppression effects. T-r1a was found to have a particularly strong suppressive effect.

[0191] <Example 8: ASO targeting the core repeat region of the SCA31 mutant repeat (TK2): MOE / DNA gapmer> (Purpose) An ASO (MOE / DNA gapmer) targeting the core repeat region of the SCA31 mutant repeat (TK2) will be synthesized to verify its effect on suppressing gene expression of the SCA31 mutant repeat (TK2).

[0192] (Method and Results) ASOs were synthesized using the same method as in Example 3. The synthesized ASOs are shown in Table 7. The position of each ASO in the SCA31 mutation repeat (TK2) is also shown in Figure 14.

[0193]

[0194] Using a method similar to that used in Example 2, ASO (0.5 nM or 2 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the TK2 direction, and the expression level of the SCA31 mutant repeat (TK2) was quantified by qRT-PCR.

[0195] The results of quantifying the expression level of the SCA31 mutant repeat (TK2) are shown in Figure 15. All of the ASOs shown in Table 7 exhibited a gene expression-suppressing effect, with T-r2'-1 exhibiting a particularly strong expression-suppressing effect.

[0196] Example 9: ASO targeting SCA31 mutant repeat (TK2): MOE / ENA / DNA gapmer (Objective) To synthesize MOE / ENA / DNA gapmers with modified positions and numbers of MOE and ENA in the wing region and verify their effect on suppressing gene expression of SCA31 mutant repeat (TK2).

[0197] (Method and Results) ASO was synthesized using the same method as in Example 3. The synthesized ASO is shown in Table 8.

[0198]

[0199] Using the same method as in Example 2, ASO (0.5 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the TK2 direction, and the expression level of the SCA31 mutant repeat (TK2) was quantified by qRT-PCR.

[0200] The results of quantifying the expression level of the SCA31 mutant repeat (TK2) are shown in Figure 16. All of the ASOs listed in Table 8 exhibited strong gene expression-suppressing effects, with T-r2'#4, T-r2'#15, T-r2'#23, T-r2'#24, and T-r2'#25 exhibiting particularly strong expression-suppressing effects. T-r2'#23 and T-r2'#25, which contain two ENAs in the 5' and 3' wing regions, exhibited particularly strong expression-suppressing effects.

[0201] Example 10: ASO targeting SCA31 mutant repeat (TK2): MOE / ENA / DNA gapmer (Objective) To synthesize MOE / ENA / DNA gapmers with modified positions and numbers of MOE and ENA in the wing region and to verify their effect on suppressing gene expression of SCA31 mutant repeat (TK2).

[0202] (Method and Results) ASO was synthesized using the same method as in Example 3. The synthesized ASO is shown in Table 9.

[0203]

[0204] Using the same method as in Example 2, ASO (0.5 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the TK2 direction, and the expression level of the SCA31 mutant repeat (TK2) was quantified by qRT-PCR.

[0205] The results of quantifying the expression level of the SCA31 mutant repeat (TK2) are shown in Figure 17. All of the ASOs shown in Table 9 exhibited strong gene expression-suppressing effects, with T-r2'#30, T-r2'#32, and T-r2'#33 exhibiting particularly strong expression-suppressing effects.

[0206] Example 11: ASO targeting SCA31 mutant repeat (TK2): MOE / ENA / DNA gapmer (Objective) To synthesize MOE / ENA / DNA gapmers with modified positions and numbers of MOE and ENA in the wing region and to verify their effect on suppressing gene expression of SCA31 mutant repeat (TK2).

[0207] (Method and Results) ASO was synthesized using the same method as in Example 3. The synthesized ASO is shown in Table 10.

[0208]

[0209] Using the same method as in Example 2, ASO (0.5 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the TK2 direction, and the expression level of the SCA31 mutant repeat (TK2) was quantified by qRT-PCR.

[0210] The results of quantifying the expression level of the SCA31 mutant repeat (TK2) are shown in Figure 18. All of the ASOs shown in Table 10 exhibited a strong expression-suppressing effect.

[0211] Example 12: ASO targeting SCA31 mutant repeat (TK2): 2'-OMe RNA / ENA / DNA gapmer (Objective) A 2'-OMe RNA / ENA / DNA gapmer containing 2'-OMe RNA and ENA within the wing region was synthesized to verify its effect of suppressing gene expression of SCA31 mutant repeat (TK2).

[0212] (Method and Results) ASO was synthesized using the same method as in Example 3. The synthesized ASO is shown in Table 11.

[0213]

[0214] Using the same method as in Example 2, ASO (0.5 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the TK2 direction, and the expression level of the SCA31 mutant repeat (TK2) was quantified by qRT-PCR.

[0215] The results of quantifying the expression level of the SCA31 mutant repeat (TK2) are shown in Figure 19. All of the ASOs shown in Table 11 exhibited strong gene expression-suppressing effects, with T-r2'#13wOme and T-r2'#23wOme exhibiting the strongest expression-suppressing effects.

[0216] Example 13: Synthesis of heteroduplex nucleic acid (HDO) targeting SCA31 mutant repeat (TK2) A heteroduplex nucleic acid (HDO) consisting of an ASO targeting the SCA31 mutant repeat (TK2) and its complementary strand was synthesized. The number of phosphorothioate bonds (PS bonds) in the ASO constituting the HDO was reduced to phosphodiester bonds.

[0217] The synthesis method of HDO will be explained using T-re10a HDO as an example.

[0218] (1) Synthesis of TK2-r2 TK2-r2 shown below: HO-C m1s -C m1s -A m1s -U rp -U rp -C rp -C rp -A rp -U rp -U rp -C rs -C m1s -A m1t -H (SEQ ID NO: 140) (the meanings of the symbols in the formula are as defined in [Chemical Formula 1] above) is synthesized.

[0219] Synthesis was carried out using an automated nucleic acid synthesizer (Gene Design nS-8 II) using the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984)). The reagents used were activator solution-3 (0.25 mol / L 5-benzylthio-1H-tetrazole in acetonitrile, Wako Pure Chemical Industries, Ltd., product No. 013-20011), CAP A for AKTA (1-methylimidazole in acetonitrile, Sigma-Aldrich, product No. L040050), Cap B1 for AKTA (acetic anhydride in acetonitrile, Sigma-Aldrich, product No. L050050), Cap B2 for AKTA (pyridine in acetonitrile, Sigma-Aldrich, product No. L050150), DCA Deblock (dichloroacetic acid in toluene, Sigma-Aldrich, product No. L023050) and Oxidizer 0.05M Iodine for AKTA (pyridine / water / iodine = 9 / 1 / 12.7 (v / v / w), Sigma-Aldrich) were used. The thiolation reagent for forming phosphorothioate bonds was phenylacetyl disulfide (CARBOSYNTH, product No. FP07495) dissolved in a 1:1 (v / v) solution of acetonitrile (anhydrous, Kanto Chemical, product No. 01837-05) and pyridine (anhydrous, Kanto Chemical, product No. 11339-05) to a concentration of 0.2 M. The amidite reagents used were RNA phosphoramidites (adenosine product No. ANP-5671, cytidine product No. ANP-6676, uridine product No. ANP-5674) and 2'-OMe nucleoside phosphoramidites (adenosine product No. ANP-5751, cytidine product No. ANP-5752) were manufactured by ChemGenes.The compound of this example was synthesized using Primer Support 5G Unylinker 350 (manufactured by GE Healthcare) as the solid support, with the time required for condensation of the amidite compound being approximately 10 minutes.

[0220] Protected oligonucleotide analogs bearing the target sequence were treated with 6 mL of concentrated aqueous ammonia / ethanol (3 / 1 v / v) to cleave the oligomer from the support and remove the cyanoethyl protecting group on the phosphorus atom and the protecting groups on the nucleobases. The resulting deprotected solution was filtered to remove the support and concentrated to dryness under reduced pressure. The resulting residue was treated with 5 mL of 1.0 M TBAF in THF (Tokyo Chemical Industry) to remove the TBDMS group on the 2'-OH of the RNA. The resulting oligomer mixture was added to 30 mL of a 0.1 M TEAB solution prepared from 1.0 M TEAB (Wako Pure Chemical Industries) and purified by reverse-phase open column chromatography (Cosmosil ODS silica gel, solution A: 0.1 M TEAB solution, solution B: acetonitrile, B%: 0% → 100% (16 min, linear gradient; 20 mL / min). The fractions containing the target product were collected and concentrated under reduced pressure. The resulting residue was treated with DOWEX 50 (Wako Pure Chemical Industries, Ltd.). The resulting solution was concentrated under reduced pressure, and 0.01 M hydrochloric acid was added and stirred at pH 2.0 for 1 hour to remove the 5'-OH protecting group. The resulting reaction solution was neutralized with concentrated aqueous ammonia. The resulting oligomer mixture was mixed with 30 mL of 0.1 M TEAB solution prepared from 1.0 M TEAB solution (Wako Pure Chemical Industries, Ltd.) and loaded onto a Clarity QSP 5 g / 6 mL Cartridge (Phenomenex, Ltd.). After adding 30 mL of 0.1 M TEAB solution, the components extracted with 30 mL of 5.0, 6.6, and 10% acetonitrile / 20 mM Tris aqueous solution were collected. The resulting oligomer solution was concentrated by ultrafiltration using an Amicon Ultra 3K (Merck, Ltd.). PBS (Thermo Fisher) was added to exchange salts, and then the mixture was desalted to obtain the target compound.This compound was analyzed by reverse-phase HPLC (column (Phenomenex, Clarity 2.6 μm Oligo-MS 100A (2.1 × 50 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM triethylamine aqueous solution, solution B: methanol, B%: 10% → 25% (4 min) → 40% (2 min) → 65% (2 min) → 100% (2 min); 60 °C; 0.5 mL / min; 260 nm) and eluted at 1.44 min. The compound was identified by negative ion electrospray ionization mass spectrometry (observed value: 4129.52).

[0221] (2) Preparation of T-re10a HDO, a double-stranded molecule composed of T-re10a (SEQ ID NO: 65) and TK2-r2 (SEQ ID NO: 140). T-re10a (2.0 μmol, SEQ ID NO: 65) and TK2-r2 (2.0 μmol, SEQ ID NO: 140) were dissolved in Milli-Q water (1000 μL) and mixed in equimolar amounts (1.8 μmol). The mixture was heated at 70°C for 5 minutes and then cooled on ice for 5 minutes to obtain a mixed solution of the two compounds. The resulting mixture was subjected to electrophoresis on a 20% polyacrylamide gel (Tris-bolate EDTA (TBE) buffer, 200 V constant voltage, 1 hour). The gel was stained and visualized with methylene blue. The bands corresponding to the single-stranded T-re10a and TK2-r2, respectively, disappeared, and a new band with a lower mobility than T-re10a and TK2-r2 was generated. The compound corresponding to the newly generated band was identified as T-re10a HDO, a double-stranded molecule formed by T-re10a and TK2-r2. T-re10a HDO was obtained by freeze-drying the mixture of the two compounds.

[0222] The nucleic acids constituting HDO synthesized by the same method as above are shown in Table 12. The structure of the nucleic acid chain constituting HDO is shown in Table 13.

[0223]

[0224]

[0225] Example 14: Verification of the gene expression suppression effect of the HDO synthesized in Example 13 (Objective) The gene expression suppression effect of the SCA31 mutation repeat (TK2) by the HDO synthesized in Example 13 was verified.

[0226] (Methods and Results) Using the same method as in Example 2, HDO (0.5 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the TK2 direction, and the expression level of the SCA31 mutant repeat (TK2) was quantified by qRT-PCR.

[0227] The results of quantifying the expression level of SCA31 mutation repeat (TK2) are shown in Figure 20. All of the HDOs shown in Table 13 exhibited a strong expression-suppressing effect.

[0228] Example 15: Mixmer targeting SCA31 mutant repeat (TK2) (Objective) A mixmer targeting SCA31 mutant repeat (TK2) was synthesized to verify the effect of suppressing gene expression of SCA31 mutant repeat (TK2).

[0229] (Method and Results) ASO was synthesized using the same method as in Example 3. The synthesized ASO is shown in Table 14.

[0230]

[0231] Using the same method as in Example 2, the ASO (2 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the TK2 direction, and the expression level of the SCA31 mutant repeat (TK2) was quantified by qRT-PCR.

[0232] The results of quantifying the expression level of the SCA31 mutant repeat (TK2) are shown in Figure 21. Among the mixmers shown in Table 14, T-r1-LNA / DNA mixmer (T-LDM) showed a good expression suppression effect.

[0233] Example 16: ASO (LNA / DNA gapmer) targeting SCA31 mutant repeat (BEAN1) An ASO (LNA / DNA gapmer) targeting the SCA31 abnormal repeat region in the mutant BEAN1 gene transcript (hereinafter referred to as "SCA31 mutant repeat (BEAN1)") was synthesized in the same manner as in Example 1. The synthesized nucleic acid is shown in Table 15 below and Figure 23.

[0234]

[0235] The SCA31 mutant repeat (BEAN1) was transiently expressed and the ASO was introduced into HeLa cells to verify its effect on gene expression. For transient expression of the SCA31 mutant repeat (BEAN1), the SCA31 mutant repeat (BEAN1) sequence was placed downstream of the chicken β-actin promoter, followed immediately by the pkSCX-IRES-EGFP vector containing an IRES-EGFP sequence (Figure 36B), as in Example 2. As in Example 2, the ASO (2 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the BEAN1 orientation, and the expression level of the SCA31 mutant repeat (BEAN1) was quantified by qRT-PCR. For qRT-PCR, a probe primer mixture: SCA31-0 (TaqMan) consisting of SCA31-0_primer FW (SEQ ID NO: 18, 5'-TGGCTGCACATAGCTTTATCTCTT-3') and SCA31-0_primer RV (SEQ ID NO: 20, 5'-AAGCCCAATCTGGAAGCAAA-3') was used.

[0236] The results of quantifying the expression level of the SCA31 mutant repeat (BEAN1) are shown in Figure 24. All compounds evaluated demonstrated strong gene expression suppression. In particular, B-r11, B-r12, B-r13, B-r14, B-r15, B-r16, B-r9, B-r10, B-r6, B-r8, B-r5, B-r4, B-r3, B-r2, B-r1, B-r7, B-j1, B-j2, B-j3, B-j4, and B-j5 exhibited strong suppressive effects on the SCA31 mutant repeat (BEAN1). These results suggest that a strong suppressive effect tends to be observed when the ASO target sequence includes the boundary between the 5' region and the core repeat region, the boundary between the core repeat region and the 3' region, and when the ASO target sequence is located within the core repeat region.

[0237] FISH similar to that used in Example 2 using an LNA-(TTCCA)5 probe confirmed that the expression of the SCA31 mutant repeat (BEAN1) was suppressed by the ASO targeting the SCA31 mutant repeat (BEAN1).

[0238] <Example 18: ASO targeting SCA31 mutant repeat (BEAN1): Surrounding sequence of B-r2> (Purpose) To synthesize an ASO consisting of the surrounding sequence of B-r2, which showed a strong expression-suppressing effect on the SCA31 mutant repeat (BEAN1) in Example 16, and to verify its effectiveness.

[0239] (Method and Results) ASO was synthesized using the same method as in Example 3. The synthesized ASO is shown in Table 16.

[0240]

[0241] Using a method similar to that used in Example 16, ASO (0.1 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the BEAN1 direction, and the expression level of the SCA31 mutant repeat (BEAN1) was quantified by qRT-PCR.

[0242] The results of quantifying the expression level of the SCA31 mutant repeat (BEAN1) are shown in Figure 25. Both B-r2a and B-r2b exhibited strong gene expression suppression effects. The suppression effect of B-r2 was greater than that of B-r2a and B-r2b.

[0243] <Example 19: ASO targeting the core repeat region of the SCA31 mutant repeat (BEAN1): MOE / DNA gapmer> (Purpose) An ASO (MOE / DNA gapmer) targeting the core repeat region of the SCA31 mutant repeat (BEAN1) will be synthesized to verify its effect of suppressing gene expression of the SCA31 mutant repeat (BEAN1).

[0244] (Method and Results) ASOs were synthesized using the same method as in Example 3. The synthesized ASOs are shown in Table 17. The position of each ASO in the SCA31 mutation repeat (BEAN1) is also shown in Figure 26.

[0245]

[0246] Using a method similar to that used in Example 16, ASO (0.1 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the BEAN1 direction, and the expression level of the SCA31 mutant repeat (BEAN1) was quantified by qRT-PCR.

[0247] The results of quantifying the expression level of the SCA31 mutant repeat (BEAN1) are shown in Figure 27. All of the ASOs shown in Table 17 showed a good expression-suppressing effect.

[0248] Example 20: Mixmer targeting SCA31 mutant repeat (BEAN1) (Objective) A mixmer targeting SCA31 mutant repeat (BEAN1) was synthesized to verify the effect of suppressing gene expression of SCA31 mutant repeat (BEAN1).

[0249] (Method and Results) ASOs were synthesized using the same method as in Example 3. The synthesized ASOs are shown in Table 18.

[0250]

[0251] Using the same method as in Example 16, ASO (2 nM) was introduced into HeLa cells together with the pkSCX-IRES-EGFP vector containing the SCA31 insertion sequence in the BEAN1 direction, and the expression level of the SCA31 mutant repeat (BEAN1) was quantified by qRT-PCR.

[0252] The results of quantifying the expression level of the SCA31 mutant repeat (BEAN1) are shown in Figure 28. Among the mixmers shown in Table 18, B-r1-LNA / DNA mixmer (B-LDM) showed a relatively good expression suppression effect.

[0253] Example 21: Verification of the effect of suppressing expression of SCA31 mutation repeats in vivo (Objective) The gene expression suppression effect of ASO / HDO on SCA31 mutation repeats (TK2) and SCA31 mutation repeats (BEAN1) was verified in vivo.

[0254] (Methods and Results) (1) Generation of SCA31 Pathological Mouse Models To examine the in vivo effects of ASO, TK2-Tg mice and BEAN1-BAC-Tg mice were generated. TK2-Tg mice were generated by introducing the same pkSCX-IRES-EGFP vector (Figure 29A) containing the SCA31 insertion sequence in the TK2 orientation used in the above examples into C57BL / 6J mice. BEAN1-BAC Tg mice were generated by introducing a BAC (Figure 29B) encompassing the patient's genomic region into C57BL / 6N mice, line D. Both Tg mice expressed the same mutated sequence as in the brains of SCA31 patients and exhibited gait abnormalities.

[0255] The TK2-Tg mice and BEAN-BAC-Tg mice used in this example are outlined in Table 19 below.

[0256]

[0257] (2) Injection into mouse ventricles. ASO / HDO was injected into the ventricles of TK2-Tg mice (passage F5, 13 weeks old, body weight 20-30 g) and BEAN1-BAC Tg mice (passage F9, 25 weeks old, body weight 20-30 g) prepared in (1).

[0258] ASO was used as a stock solution in 50 μg / μL PBS, denatured at 85°C for 3 minutes, and then used. HDO was prepared by denaturing the same amount of cRNA as the backbone at 95°C for 5 minutes and 37°C for 60 minutes. ASO was diluted with PBS, and 10 μL was used as the drug solution. HDO was diluted with PBS, and 10 μL was used as the drug solution. 10 μL of PBS was administered as a control.

[0259] Mice were anesthetized with 2% isoflurane and maintained at 1.5-2.5% during surgery. The mice were kept warm at 38°C during and after surgery.

[0260] Injections were performed into the left lateral ventricle of mice. The injection site was 1 mm lateral and 0.2 mm caudal to the Bregma, 3 mm into the skull (indicated by a circle in Figure 30). After puncture and leaving the needle for 2 minutes, 10 μL of the drug was injected over 3 minutes, and the needle was removed after 5 minutes.

[0261] The ASOs and HDOs used in this example are shown in Tables 20 to 23, respectively.

[0262]

[0263]

[0264]

[0265]

[0266] (3) Expression Analysis by qRT-PCR. One week after ventricular injection, the bilateral hippocampi and cerebellum were removed. RNA was extracted using Trizol and then recovered by isopropanol precipitation. cDNA was prepared from 1 μg of recovered RNA using PrimeScript Takara. The product obtained from TK2-Tg mice using EASY Dilution was diluted 1:10, and the product obtained from BEAN1-BAC-Tg mice was diluted 1:5 as a template. q-PCR was performed using a Roche Light Cycler 480II. For q-PCR, the SCA31-1 (TaqMan) primers, consisting of SCA31-1_primer FW (SEQ ID NO: 10, 5'-TGGATAGAAACCCCAGCTTCCT-3') and SCA31-1_primer RV (SEQ ID NO: 15, 5'-CTGCCTCCGACACTTTTCAATT-3'), were used for TK2-Tg mice. For BEAN1 BAC-Tg mice, the AB472391-1 (Ex2-3) primers, consisting of Ex2-3_primer FW (SEQ ID NO: 25, 5'-CAGCCACTGCCCAGGACTAA-3') and Ex2-3_primer RV (SEQ ID NO: 30, 5'-GCCACTTCTCTGCCTCAGAGA-3'), were used. The positions detected by q-PCR are shown as "SCA31-1" and "EX2-3" in Figure 29. ACTB (Mm00607939_s1) was used as a reference.

[0267] The results of suppression of expression of the SCA31 mutant repeat (TK2) by T-re10a ASO (100 μg) are shown in Figure 31A. It was found that T-re10a ASO had a strong suppressive effect on the SCA31 mutant repeat in vivo.

[0268] Figure 31B shows the results of suppression of SCA31 mutant repeat (TK2) expression by T-re10a HDO (0.3 μg to 300 μg). T-re10a HDO was found to have a concentration-dependent, effective suppression of SCA31 mutant repeat expression in vivo. The effect was particularly strong at doses of 10 μg or higher.

[0269] The results of suppression of SCA31 mutant repeat (TK2) expression by T-r2'_1 ASO (100 μg) are shown in Figure 32A. Although the suppression effect was weaker than that of T-re10a ASO, T-r2'_1 ASO was also found to suppress SCA31 mutant repeat expression in vivo.

[0270] The results of suppression of SCA31 mutant repeat (BEAN1) expression by B-r2 ASO (10 μg or 50 μg) are shown in Figure 32B. Administration of 50 μg of B-r2 ASO resulted in a strong suppression of SCA31 mutant repeat (BEAN1) expression.

[0271] (4) Expression Analysis by Northern Blot RNA samples obtained from the left cerebellum in the same manner as in (3) were treated with DNase and then analyzed using Oligotex (trademark)-dt30 <super>Northern blot analysis was performed on PolyA-purified samples using the Takara mRNA Purification Kit. Electrophoresis was performed at 50 V for 3.5 hours, followed by transfer for 20 hours. The RNA was then immobilized on the membrane by UV cross-linking. Hybridization was performed overnight at 45°C using DIG-labeled LNA-(TTCCA)5 probe (SEQ ID NO: 31) and LNA-(TGGAA)5 probe (SEQ ID NO: 32), followed by detection with an anti-DIG-AP antibody. The DIG Wash and Block Buffer Set was used, and color development was performed with CDP Star.

[0272] The Northern blot results are shown in Figure 31C. In Figure 31C, the first three lanes from the left show the results of SCA31 repeat detection using 1 μg, 0.5 μg, and 0.25 μg RNA samples obtained from PBS-injected negative control mice. The fourth lane from the left shows the results of SCA31 repeat detection using 1 μg RNA sample obtained from mice injected with 300 μg of T-re10a HDO. The results show that administration of T-re10a HDO reduced the expression of the SCA31 mutant repeat (TK2) by more than four-fold.

[0273] Example 22: HDO containing 2'-O-MOE-RNA nucleoside (Objective) An HDO containing T-re10a as the first nucleic acid strand and 2'-O-MOE-RNA nucleoside as the second nucleic acid strand is prepared, and its effect of suppressing gene expression of the SCA31 mutant repeat (TK2) is verified.

[0274] (Method and Results) HDO was prepared in the same manner as in Example 21. The prepared HDO is shown in Table 24 below.

[0275]

[0276] Using the same method as in Example 21, HDO was injected into the cerebral ventricles of TK2-Tg mice, and one week later, the expression of the SCA31 mutant repeat (TK2) in the hippocampus and cerebellum of both sides was analyzed by qRT-PCR.

[0277] The results of suppression of SCA31 mutant repeat (TK2) expression by T-re10a HDO (Default), T-re10a HDO (cMOE / DNA), T-re10a HDO (Full cMOE), T-re10a HDO (cMOE / DNA_2), and T-re10a HDO (cMOE / DNA_3) are shown in Figure 34. Each HDO was found to have a strong suppressive effect on SCA31 mutant repeat expression in vivo.

[0278] The results of suppression of SCA31 mutant repeat (TK2) expression by administration of 100 μg of T-re10a HDO (Default), 100 μg, 10 μg, or 1 μg of T-re10a HDO (cMOE / DNA), and 100 μg, 10 μg, or 1 μg of T-re10a HDO (Full cMOE) are shown in Figures 35 and 37. Increasing the dose of T-re10a HDO (cMOE / DNA) and T-re10a HDO (Full cMOE) demonstrated an increased suppression effect on SCA31 mutant repeat (TK2). All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.< / super>

Claims

1. A nucleic acid molecule having an antisense effect on a transcript of a mutant gene containing an abnormal repeat region, the abnormal repeat region consists of a 5' region, a core repeat region, and a 3' region; the core repeat region consists of a base sequence in which the base sequence TGGAA is repeated multiple times, or a base sequence complementary thereto; The nucleic acid molecule comprises a base sequence complementary to four or more consecutive bases in the abnormal repeat region of the transcript, and comprises a target binding region capable of hybridizing to the transcript.

2. The nucleic acid molecule of claim 1, wherein the mutant gene is a mutant NEDD4-associated brain-expressed 1 (BEAN1) gene, the core repeat region consists of a base sequence in which the base sequence TGGAA is repeated multiple times, and the 5' region contains the base sequence TCAC.

3. The nucleic acid molecule according to claim 2, wherein the 5' region consists of any one of the base sequences selected from the group consisting of (a1) to (a4) below: (a1) the base sequence shown in SEQ ID NO: 3; (a2) the base sequence shown in SEQ ID NO: 4; (a3) the base sequence TCAC, and (a4) A base sequence formed by linking the base sequence TCAC and a base sequence in which the base sequence TAGAA is repeated multiple times in order from the 5' end.

4. The nucleic acid molecule of claim 1, wherein the mutant gene is a mutant NEDD4-associated brain-expressed 1 (BEAN1) gene, the core repeat region consists of a base sequence in which the base sequence TGGAA is repeated multiple times, and the 3' region includes a sequence in which the base sequence TAGAA is repeated multiple times and a sequence in which the base sequence shown in sequence number 6 is repeated multiple times.

5. The nucleic acid molecule according to claim 4, wherein the 3' region consists of any one of the base sequences selected from the group consisting of (b1) to (b5) below: (b1) a base sequence in which the base sequence shown in SEQ ID NO: 7, a base sequence in which the base sequence TAGAA is repeated multiple times, and a base sequence in which the base sequence shown in SEQ ID NO: 6 is repeated multiple times are linked in order from the 5' end; (b2) a base sequence in which the base sequence shown in SEQ ID NO: 9, a base sequence in which the base sequence TAGAA is repeated multiple times, and a base sequence in which the base sequence shown in SEQ ID NO: 6 is repeated multiple times are linked in order from the 5' side; (b3) a base sequence in which the base sequence shown in SEQ ID NO: 11, a base sequence in which the base sequence TAGAA is repeated multiple times, and a base sequence in which the base sequence shown in SEQ ID NO: 6 is repeated multiple times are linked in order from the 5' side; (b4) a base sequence in which the base sequence shown in SEQ ID NO: 12, the base sequence in which the base sequence TAGAA is repeated multiple times, the base sequence in which the base sequence shown in SEQ ID NO: 6 is repeated multiple times, and the base sequence shown in SEQ ID NO: 13 are linked in order from the 5' end; and (b5) A base sequence in which the base sequence shown in SEQ ID NO: 14, a base sequence in which the base sequence TAGAA is repeated multiple times, and a base sequence in which the base sequence shown in SEQ ID NO: 6 is repeated multiple times are linked in order from the 5' end.

6. 3. The nucleic acid molecule of claim 2, wherein the target binding region comprises a non-mutated region adjacent to the 5' side of the 5' region, the 5' region, the core repeat region, the 3' region, and bases complementary to the terminal bases of two adjacent regions in the non-mutated region adjacent to the 3' side of the 3' region.

7. The nucleic acid molecule of claim 2 , wherein the entire length of the target binding region is contained in a base sequence complementary to either the core repeat region or the 3′ region.

8. The nucleic acid molecule of claim 2 , wherein the entire length of the target binding region is contained in a base sequence complementary to the core repeat region.

9. The nucleic acid molecule according to claim 2, consisting of any one of the base sequences selected from the group consisting of SEQ ID NOs: 156 to 189 and 207 to 209 (wherein t may be u).

10. The nucleic acid molecule of claim 1, wherein the mutant gene is a mutant thymidine kinase 2 (TK2) gene, the core repeat region consists of a base sequence complementary to a base sequence in which the base sequence TGGAA is repeated multiple times, and the 5' region comprises a sequence in which the base sequence shown in SEQ ID NO: 16 is repeated multiple times and a sequence in which the base sequence TTCTA is repeated multiple times.

11. The nucleic acid molecule according to claim 10, wherein the 5' region consists of any one of the base sequences selected from the group consisting of (c1) to (c5) below: (c1) a base sequence in which the base sequence shown in SEQ ID NO: 16 is repeated multiple times, a base sequence in which the base sequence TTCTA is repeated multiple times, and a base sequence in which the base sequence shown in SEQ ID NO: 19 is linked in this order from the 5' end; (c2) a base sequence in which the base sequence shown in SEQ ID NO: 16 is repeated multiple times, a base sequence in which the base sequence TTCTA is repeated multiple times, and a base sequence in which the base sequence shown in SEQ ID NO: 21 is linked in this order from the 5' end; (c3) a base sequence in which the base sequence shown in SEQ ID NO: 16 is repeated multiple times, a base sequence in which the base sequence TTCTA is repeated multiple times, and a base sequence in which the base sequence shown in SEQ ID NO: 22 is linked in this order from the 5' end; (c4) a base sequence in which the base sequence shown in SEQ ID NO: 23, the base sequence shown in SEQ ID NO: 16 are repeated multiple times, the base sequence in which the base sequence TTCTA is repeated multiple times, and the base sequence shown in SEQ ID NO: 24 are linked in order from the 5' end; and (c5) A base sequence in which the base sequence shown in SEQ ID NO: 16 is repeated multiple times, a base sequence in which the base sequence TTCTA is repeated multiple times, and a base sequence in which the base sequence shown in SEQ ID NO: 29 is linked in order from the 5' end.

12. The nucleic acid molecule of claim 1, wherein the mutant gene is a mutant thymidine kinase 2 (TK2) gene, the core repeat region consists of a base sequence complementary to a base sequence in which the base sequence TGGAA is repeated multiple times, and the 3' region contains the base sequence GTGA.

13. The nucleic acid molecule according to claim 12, wherein the 3' region consists of any one of the base sequences selected from the group consisting of (d1) to (d4) below: (d1) the base sequence shown in SEQ ID NO: 27; (d2) the base sequence shown in SEQ ID NO: 28; (d3) the base sequence GTGA, and (d4) A base sequence in which the base sequence TTCTA is repeated multiple times and the base sequence GTGA are linked in order from the 5' end.

14. 11. The nucleic acid molecule of claim 10, wherein the target binding region comprises bases complementary to the terminal bases of the 5' region, the core repeat region, the 3' region, and two adjacent regions in the non-mutated region adjacent to the 3' side of the 3' region.

15. The nucleic acid molecule of claim 10 , wherein the entire length of the target binding region is contained in a base sequence complementary to either the 5′ region or the core repeat region.

16. The nucleic acid molecule of claim 10 , wherein the entire length of the target binding region is contained in a base sequence complementary to the core repeat region.

17. The nucleic acid molecule according to claim 10, consisting of any one of the base sequences selected from the group consisting of SEQ ID NOs: 33 to 53, 58 to 139, 141 to 150, 152 to 155, and 204 to 206 (provided that t in the sequence may be u).

18. The nucleic acid molecule of claim 1, wherein the mutant gene is a mutant ataxin 10 (ATXN10) gene, the core repeat region consists of a base sequence complementary to a base sequence in which the base sequence TGGAA is repeated multiple times, and the entire length of the target binding region is contained in the base sequence complementary to the core repeat region.

19. The nucleic acid molecule according to claim 18, consisting of any one of the base sequences selected from the group consisting of SEQ ID NOs: 33 to 37, 58 to 61, 64 to 67, 70 to 139, 141 to 150, 152 to 155, and 204 to 206 (however, t in the sequence may be u).

20. The nucleic acid molecule of claim 1, which is 12 to 30 bases in length.

21. The nucleic acid molecule of claim 1 which is a mixmer.

22. The nucleic acid molecule of claim 1 which is a gapmer.

23. (1) a central region containing at least two consecutive deoxyribonucleosides; (2) a 5' wing region including an unnatural nucleoside, located on the 5' end of the central region; and (3) a 3' wing region containing an unnatural nucleoside located on the 3' end side of the central region;

24. 24. The nucleic acid molecule of claim 23, wherein the 5' wing region and the 3' wing region comprise bridged nucleosides and / or 2' modified nucleosides.

25. 25. The nucleic acid molecule of claim 24, wherein the bridged nucleoside is an LNA nucleoside or an ENA nucleoside.

26. 25. The nucleic acid molecule of claim 24, wherein the 2'-modified group of the 2'-modified nucleoside is a 2'-O-methyl group or a 2'-O-methoxyethyl group.

27. The nucleic acid molecule of claim 1, comprising or consisting of a morpholino nucleic acid.

28. 2. The nucleic acid molecule of claim 1, wherein all or some of the internucleoside linkages of the nucleic acid molecule are modified internucleoside linkages.

29. 29. The nucleic acid molecule of claim 28, wherein the modified internucleoside linkage is a phosphorothioate linkage.

30. The nucleic acid molecule of claim 1, comprising a modified nucleobase.

31. The nucleic acid molecule of claim 1 , wherein the antisense effect is a reduction in the amount of the transcript.

32. The nucleic acid molecule of claim 1 , wherein the antisense effect is a steric block.

33. A double-stranded nucleic acid complex comprising a first nucleic acid strand consisting of the nucleic acid molecule of claim 1 and a second nucleic acid strand comprising a base sequence complementary to the first nucleic acid strand.

34. 34. The double-stranded nucleic acid complex of claim 33, wherein the second nucleic acid strand comprises ribonucleosides, deoxyribonucleosides, and / or modified nucleosides.

35. In the second nucleic acid strand, all nucleosides in a region consisting of a base sequence complementary to the central region of the first nucleic acid strand are (a) deoxyribonucleosides, (b) deoxyribonucleosides and ribonucleosides, (c) deoxyribonucleosides and 2'-modified nucleosides, (d) ribonucleosides and 2'-modified nucleosides, or (e) Deoxyribonucleosides, ribonucleosides, and 2'-modified nucleosides The double-stranded nucleic acid complex of claim 33, wherein

36. A nucleic acid molecule according to claim 23, comprising a first nucleic acid strand and a second nucleic acid strand comprising a base sequence complementary to the first nucleic acid strand, A double-stranded nucleic acid complex, wherein the second nucleic acid strand comprises a region containing at least two consecutive ribonucleosides and / or deoxyribonucleosides complementary to at least two consecutive deoxyribonucleosides in the central region of the first nucleic acid strand.

37. The double-stranded nucleic acid complex of claim 36, wherein the second nucleic acid strand contains a modified internucleoside linkage in a region consisting of a base sequence complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand.

38. 38. The double-stranded nucleic acid complex of claim 37, wherein the modified internucleoside linkage is a phosphorothioate linkage.

39. The double-stranded nucleic acid complex of claim 36, wherein the second nucleic acid strand contains bridged nucleosides and / or 2'-modified nucleosides in a region consisting of a base sequence complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand.

40. In the second nucleic acid strand, the bridged nucleoside is an LNA nucleoside, an ENA nucleoside, or a BNA NC 40. The double-stranded nucleic acid complex of claim 39, which is a nucleoside.

41. 40. The double-stranded nucleic acid complex of claim 39, wherein in the second nucleic acid strand, the 2'-modified group of the 2'-modified nucleoside is a 2'-O-methyl group or a 2'-O-methoxyethyl group.

42. 34. The double-stranded nucleic acid complex of claim 33, wherein the second nucleic acid strand comprises one or more 2'-O-methoxyethyl modified nucleosides.

43. 43. The double-stranded nucleic acid complex of claim 42, wherein at least 20% of the total number of nucleosides in the second nucleic acid strand are 2'-O-methoxyethyl modified nucleosides.

44. The double-stranded nucleic acid complex of claim 33, wherein the second nucleic acid strand comprises one or two or more consecutive 2'-O-methoxyethyl modified nucleosides located at the 5'-terminus and / or one or two or more consecutive 2'-O-methoxyethyl modified nucleosides located at the 3'-terminus.

45. The double-stranded nucleic acid complex of claim 33, wherein the second nucleic acid strand comprises 1 to 7 2'-O-methoxyethyl modified nucleosides at a position other than the 5' end and the 3' end.

46. 34. The double-stranded nucleic acid complex of claim 33, wherein in the second nucleic acid strand, all nucleosides other than 2'-O-methoxyethyl modified nucleosides are deoxyribonucleosides.

47. 34. The double-stranded nucleic acid complex of claim 33, wherein all of the nucleosides in the second nucleic acid strand are 2'-O-methoxyethyl modified nucleosides.

48. 34. The double-stranded nucleic acid complex of claim 33, wherein the second nucleic acid strand comprises a modified nucleobase.

49. 34. The double-stranded nucleic acid complex of claim 33, wherein the second nucleic acid strand is bound to tocopherol or cholesterol, or an analog thereof.

50. the base sequence of the first nucleic acid strand consists of the base sequence set forth in SEQ ID NO: 65 (wherein t may be u); and The double-stranded nucleic acid complex of claim 33, wherein the base sequence of the second nucleic acid strand consists of any one of the base sequences selected from the group consisting of SEQ ID NO: 140, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, and SEQ ID NO: 203 (provided that t in the sequence may be u).

51. the first nucleic acid strand consists of the nucleic acid strand set forth in SEQ ID NO: 65; and 51. The double-stranded nucleic acid complex of claim 50, wherein the second nucleic acid strand consists of any one of the nucleic acid strands selected from the group consisting of SEQ ID NO: 140, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, and SEQ ID NO:

203.

52. A nucleic acid molecule according to any one of claims 2 to 9 and claims 20 to 32 which cite them, or a double-stranded nucleic acid complex according to any one of claims 33 to 49 which cite them, and / or The nucleic acid molecule according to any one of claims 10 to 19 and claims 20 to 32 which cite them, or the double-stranded nucleic acid complex according to any one of claims 33 to 51 which cite them. A pharmaceutical composition for treating spinocerebellar ataxia type 31 (SCA31), comprising:

53. A pharmaceutical composition for treating spinocerebellar ataxia type 10 (SCA10), comprising the nucleic acid molecule according to claim 18 or 19, or any one of claims 20 to 32 quoting thereto, or the double-stranded nucleic acid complex according to any one of claims 33 to 51 quoting thereto.

54. 53. The pharmaceutical composition of claim 52, which is administered intracerebroventricularly or intrathecally.

55. 53. The pharmaceutical composition of claim 52, wherein the single dose of the nucleic acid molecule is 0.1 mg / kg or more.